Staining Methods. PAUL I. HOElER, Inc. Medical Division of Harper & Brothers

Transcription

1 Staining Methods PAUL I. HOElER, Inc. Medical Division of Harper & Brothers

2 HistologiC and Histochemical J. F. A. McMANUS, M.D. Professor and Chairman of Department of Pathology University of Alabama Medical Center ROBERT W. MOWRY, M.D. Professor of Pathology University of Alabama Medical Center

3 For A. E. J. and T. B. T. Staining Methods Histologic and Histochemical Copyright 1960, by Paul B. Roeber, Inc, Medical Division of Harper & Brothers Prmted in the United States of America All rights reserved. For information address Paul B. Hoeber, Inc, MedIcal DIvIsion of Harper & Brothers, 49 East 33rd Street, New York 16, N Y. Published September, 1960 H-K LIbrary of Congress catalog card number:

4 Contents PREFACE vii I. DIRECT EXAMINATION OF TISSUES AND PREPARATION OF TISSUE SECTIONS AND SMEARS 1. Unfixed Tissues 3 2. Tissue Fixation and Commonly Used Fixatives of Wide Utility 8 3. Fixed Tissues Tissue Sectioning Treatment of Sections Before and After Staining 47 II. III. METHODS OF GENERAL UTILITY FOR THE ROUTINE STUDY OF TISSUES 6. Paraffin Section Methods Frozen Section Routines in Surgical Pathology 65 GENERAL METHODS FOR STUDY OF THE CELL AND ITS STRUCTURES 8. The Nucleus The Cytoplasm Secretory and Storage Material 90 IV. SPECIAL METHODS FOR THE CONSTITUENTS OF CELL AND TISSUES 11. Proteins and Related Substances Fats and Fatlike Substances Carbohydrates Enzymes Inorganic Elements; Pigments; Foreign Substances Extracellular Materials: Fibrous Components Extracellular Materials: Nonfibrous Components 246 v

5 vi Contents V. THE STUDY OF SPECIAL CELLS, TISSUES, AND ORGANS 18. Connective Tissues 19. Blood and Blood-Forming Organs 20. Skin, Mucous Membranes, and Breast 21. Musculoskeletal System 22. Respiratory System 23. Cardiovascular System 24. Digestive Syster:n 25. Genitourinary Tract 26. Endocrine Glands 27. Nervous System 28. Organs of Special Sense 29. Pregnancy and Products of Conception 30. Demonstration of Bacteria, Fungi, and Other Parasites in Tissue Sections Appendix A. Appendix B. BIBLIOGRAPHY INDEX Outline of Basic Techniques for Study of Various Organs and Systems 375 Dilution and Solubility Tables; Molar Values; Buffers Used in Histochemistry

6 Preface The improvement of histologic techniques and the multiplication of histochemical methods have created the need for a handbook integrating the newer methods of tissue examination into the standard laboratory procedures. This book was prepared to fill that need. Therefore, the authors selected from the perplexing array available the single or several methods they considered most valuable for the adequate and efficient staining of hist?logic preparations. Those presented are regarded as effective, reliable, and easily mastered; they are largely those with which the authors have had a considerable amount of personal experience. In addition to staining methods, the book includes a discussion of the essential processes for preparation of the tissues for staining: fixation, dehydration, embedding, and sectioning. Also included is a special section of recommendations concerning methods especially useful for the study of specific body organs and tissues. The range of procedures and stains covered ensures that the book will be useful in routine work and in research by pathologists, histologists, histochemists, cytologists, and their technical associates. The systematic organization of the contents, which facilitates study as well as locating a particular procedure, enables the novice in the techniques of histology and histochemistry to use the book. It is hoped that it will prove valuable to anyone who examines human or animal tissue with the microscope. Although this book was mutually planned, circumstances restricted the contribution of Dr. Mowry to specific areas of carbohydrate histochemistry and to the discussion of sulfation and other methods. However, his frequent discussions of many other aspects of the work with the senior author have been extremely valuable. A book such as this is necessarily derivative, being built upon the experience of earlier authors. We wish to make a special expression of our debt to the older workers in the field: especially to Mallory, to Bertrand, to Ramon y Cajal, through whose books we first were led into the study of tissues and cells. Our debt is no less to more recent authors: Lillie, Pearse, Comori, and Click; to Carleton and to Langeron. The late W. C. MacCallum demonstrated to one of us (McM.) the potentialities of information that could come from a study of cells and tissues and John Baker, fifteen years ago, introduced him to histochemistry. Each of vii

7 viii Preface these individuals should be given credit for what is worth while in this treatise while being in no way culpable for any deficiencies. A definite but largely imponderable participation in the making of this book has been that by our various technicians. S. Sgt. Hugh MItchell, RCAMC; Mrs. Sara Howell DeWitt; Mrs. Jane Cason Simpson; MISS Eunice Johnson; MIss Eoline McGowan, Mrs. Ann Clancey Daly; Mrs. Delores Baker Madden; Mrs. Florence Siegel; Miss Angelina Giovino; Miss Mildred MacCaulley. The secretarial duties have been performed by Mrs. William Wenger, with the help of Mrs. Troy Cross and Mrs. Robert Barnes; to them we are sincerely grateful. Many of our associates' are listed in the bibliography in jomt publications. Their contribution cannot be overestimated, Helpful- stimulation and suggestions have come from Drs. C. H. Lupton, S. P. Kent, J. A. Cunningham, J. C. Saunders, G. B. Penton, R. H. Jordan, William Boyd, F. C. D. Collier, C. O. and B. M. Hathaway, and others. Birmingham, Alabama J. F. A. McM.

8 Section I DIRECT EXAMINATION OF TISSUES AND PREPARATION OF TISSUE SECTIONS AND SMEARS

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10 Unfixed Tissues DIRECT EXAMINATION Many thin membranes such as the pia arachnoid or pleura can be examined directly with the microscope even though unstained. One uses either direct or transmitted light, or a combination of both. A piece of the thin tissue, about 1 X 2 em., is cut WIth scissors or scalpel and flattened out on the slide. Cover with a drop of normal saline solution or glycerine and apply the coverslip. Many tissue details can be made out. When Illumination is reduced by rackmg down the condenser or closing the iris diaphragm to a satisfactory degree, greater contrast is obtamed. The value of staining is seen when the coverslip is removed from such a preparation, a drop or two of polychrome methylene blue (p. 67) or toluidine blue (p. 132) solution is applied, and the coverslip replaced. Under the microscope, considerably greater contrast and cellular and tissue detail can now be seen. Suitable for this kind of study are the pia arachnoid of the spinal cord and brain, thm portions of the mesentery, or stripped-off portions of the pleura, pericardium, or peritoneum. The intimal endothelium of large blood vessels can be stripped off and Similarly prepared. Mter staining with one of the dyes mentioned above, the cement substance of the endothelial cells and various granules in cells, "mcluding mast cells, are colored red or metachromatic. In contrast, the nuclei and general cytoplasm are blue. Also, a fat (p. 110) or silver stain (p. 295) can be applied to the unfixed intima or other thin membrane. Almost any histochemical and histologic procedure can be performed on these thin membranes of the body. Such unfixed membranes can be handled as frozen sections (p 5) and transferred from solution to solution, supported by glass rods or mounted on glass slides. This allows the beginner to perform various staining and histochemical procedures without going to the trouble of learning how to embed tissues in paraffin, cut sections, and so on. 3

11 4 Examination of Tissues-Preparing Tissue Sections and Smears SMEARS AND SQUASH PREPARATIONS Considerable information can be obtamed from microscopic examination of cells either smeared lightly on a slide or squashed in the botanical fashion. For the latter, choose a very small piece of tissue, perhaps 1 mm. or so in greatest dimension. Place the tissue on a slide and forcibly apply a coverslip. Frequently, enough dissociation of the tissue results that tissue details and, sometimes, cellular details are revealed. Such squash preparations can be stained by applying a drop of dye at the edge of the coverslip, the stain moving over the tissue by capillary action. Suitable for this purpose are Giemsa's stain (p. 270), polychrome methylene blue (p. 67), or 0.1 per cent aqueous toluidine blue. "Squash" preparations are used relatively little at the present time and find their greatest value in the study of friable material such as lymphoid tissue, sple~n, bone marrow, and cellular tumors. ') TOUCH PREPARATIONS If a clean glass microscopic slide is pressed against the freshly cut surface of a tissue, a certain number of cells will adhere to the surface of the slide. Such cells can be examined either unstained or after staining with toluidine blue or polychrome methylene blue; on the other hand, the touch preparation can be fixed and stained by most of the methods to be described later. FREEHAND SECTIONING Freehand sectioning can provide useful preparations of some tissues. For several hundred years before the introduction of the microtome, microscopists used only freehand sections. While the method is now little used because of the ease of frozen sectioning (p. 5), there are several situations in which freehand sectioning can be very valuable. In the study of pulmonary embolism due to fat, for example, a freehand section of the lung 1 to 3 mm. thick can be cleared in 1 per cent i;o 10 per cent potassium hydroxide, washed, and then stained with one of the Sudan dyes for fat. The section is then mounted on a slide and observed again by a combination of direct and transmitted light, using the lower powers of the microscope. Also, some information may be obtained 'about the extent and type of fibrosis in pathologic tissues by the study of sections cut this way. A piece of tissue is grasped between the thumb and forefinger and the knife is carried across it several hmes in rapid succession, parallel with and as close to the upper surface as possible. The sections are delivered into normal saline solution. Of the number cut, a few will be found thin enough to be worth microscopic exammation, either in the unstained

12 Unfixed Tissues 5 condition or with the use of the simpler staining solutions previously mentioned. The motion used in cutting such freehand sections IS very much like that of the freezing microtome. It seems probable that the former led to the knife arrangement used in the freezing microtome. -FROZEN SECTIONS Frozen sections can be produced easily without prior fixation from tissue that has a moderate degree of firmness. Such fnable tissues as the lymphoid, spleen, and very cellular tumors make poor frozen sections without prior fixation. Firmer tissues such as heart, liver, breast, and fibrous tumors are much more SUItable for frozen sectioning even when unfixed. To cut frozen ~ sections, a block of the tissue (2 X 1 X 0.4 cm.) to be sectioned is moistened by dipping it briefly in saline solution and is then placed flat on the stage of the microtome. Hold the tissue firmly against the stage with the finger while bursts of carbon dioxide under pressure are allowed to pass close to and under the stage of the microtome. The cooling effect of the carbon dioxide being released under pressure is transmitted to the stage of the microtome and, in tum, to the tissue. This produces an icy seal between the moistened tissue and the microtome. RemOVing the finger, the carbon dioxide is released, again in short bursts, until the whole tissue is frozen. This is marked by progressive paling of the tissue, owing to the formation of ice. Freezing is complete when the whole tissue is white. Satisfactory sections cannot be cut from this completely frozen tissue until slight thawing has occurred on the surface of the specimen. This is the phase in which good sectioys can be obtained. However, if the knife is kept very cool (as in some versions of the cryostat or by special attachments that provide cooling of the knife in the routine freezing microtome), sections can be cut while the tissue is still entirely frozen; but the more usual procedure follows. To encourage thawing, one's finger is dipped into water and a waterdrop is transferred by the finger to the surface of the frozen tissue. Usually several drops and momentary pressure of the finger on the tissue are required. When thawmg of the superficial portion of the tissue has begun, the knife blade is swept several times across the top of the tissue. The sections initially obtained will be too thick but as additional cuts are made, suitably thin sections will appear. Sections are not ordinarily cut much thinner than 10 microns. The upper part of the frozen-section blade should be moistened during cutting with some drops of water. As the section is cut off the thawing surface of the block, it passes onto the knife where the film of water is already present. This moistening of the knife blade prevents the often troublesome adherence of the tissue section to the blade.

13 6 Examination of Tissues-Preparing Tissue Sections and Smears The sections are removed from the blade one at a time with a damp camel's hair brush or with the outer edge of the little finger. Use a gentle sweeping motion that touches and carries the sections toward the cutting edge of the blade. The sections are gently swept off the blade by a motion that is the reverse of their movement during cuttmg. The sections are then Hoated off the brush or finger into a wide dish containing normal saline solution or some other osmotically correct solution. The use of plain water is perhaps more frequent but causes swelling of cells and tissues. It is well to cut a number of sections and then select those that appear more nearly perfect. The remainmg steps in the procedure must accomplish staining of the section and mountmg of the section on a glass slide in a manner suitable for microscopic study. There are several choices available to the worker, mainly whether to stain the section while unattached to the slide and later to mount It or whether to mount the section on the slide before staining. The most direct and probably most Widely practiced method is to use a glass rod, probe, or angular dissecting needle to carry the unmounted section through the stain and other solutions before, finally, mounting the section on the glass slide. As the stain is ordmarily opaque, it is safer to hold the section during staining on such a glass rod rather than take the chance of losing the section in the staining dish. As saline will tend to corrode metal, a glass rod of a diameter from a few millimeters to 1 cm. will prove convenient. From the saline, the tissue section is lifted by the glass rod and kept clinging to the rod as the tissue is transferred through various solutions. The portion of the section touching the glass rod can be varied by slowly rotating the rod from time to time. This maneuver allows more even staining of the entire tissue section. The stained frozen section is taken from the staining solution and excess stain is washed off by immersing it in water or sahne. Until one has had much practice, it may be difficult to Hatten out the section. (This can be more quickly overcome in the following fashion. Transferring a section from weak alcohol (30 per cent to 50 per cent) to a bath of fresh water produces a change in surface tension that causes the section to Hatten out on the surface of the water.) After the section has been Hattened, drain the slide and wipe off any excess water around the section. The coverslip may be applied to the wet tissue directly or, preferably, after application of a drop or two of an aqueous mountant such as glycerin, glycerin jelly, or others (p. 47). If the worker prefers to mount the unstained section on a glass slide before staining, he will have the convenience of being able to handle the section by means of the slide. Also, it often seems that more perfect Hattening of the section on the slide can be obtained before the section has been affected by the staining solutions. The section is Hoated onto the

14 Unfixed Tissues 7 glass slide by passing the slide beneath and at a slight angle to the surface of 'the water. Using the edge of the glass rod or bent end of a metal rod, anchor the main part of the section gently to the slide while it is removed slowly from the water. Drain the excess water off the slide and wipe around the section, taking care not to injure the tissue., Should there be objectionable wrinkles in the section when, the slide is removed from the water, these can be eliminated in the following manner: lower the edge of the glass slide corresponding to the wrinkled edge of the section so that the section is perpendicular to the water's edge. On contact with the water, the wrinkled edge of the section can usually be. flattened out by alternately immersing and withdrawing the section. In tum, other edges of the section can be similarly treated. Utmost proficiency in the flattening of sections will require much experience and patience but can be accomplished. Although the section is now flattened on the slide, it WIll not stick to the glass slide during staining unless some treatment that fixes the tissue proteins is used. With a medicine dropper or pipette, apply successive drops of 95 per cent or absolute alcohol or other fixing fluid to the surface of the section, held parallel to the working surface of the table. This will remove most of the tissue water, partly coagulate the tissue proteins, and facilitate adherence of the section to the glass slide. 'Many workers like to blow on the section between applications of fixing fluid or alcohol, either with the breath or with a small stream of compressed air from a pump. The section should now be allowed to dry partly but not excessively by placing it on top of a warm microscope lamp or beneath a light bulb. The slide bearing the section can now be either stained directly or given an additional treatment of a few minutes or so in 95 per cent alcohol and then stained. The method of mounting the section on the slide has the advantage that a number of slides can be stained and handled at the same time When the section is being held on a glass rod, it is difficult to process more than one section at a time. Unfortunately, frozen sections of fixed tissues do not adhere very consistently to glass slides treated by the method just described. For certain purposes, for example, when large numbers of frozen sections are being stained at one time, one may float them into a small paper cup whose bottom is perforated with numerous tiny pinpricks. The sections can be carried from one staining solution to other baths merely by transferring the paper cup and successively immersing the bottom beneath the fluid level of each bath. In the final step, allow the sections to float out into a wide dish full of water and mount them in the usual manner. In surgical pathology, frozen sections stained with hematoxylin and eosin possess certain advantages for rapid diagnosis. The procedure in use at the UniversIty of Alabama Medical Center is as detailed on page 67.

15 2 Tissue Fixation and Commonly Used Fixatives of Wide Utility PURPOSES OF FIXATION The general distribution of materials making up cell structure visible by light microscopy should be preserved by adequate fixation. Furthermore, the distribution should not be affected substantially by subsequent -dehydrating, embedding, and sectioning procedures. Fmally, cell structures should still be susceptible of absorbmg contrast-producmg materials such as stains, metals, and colored products of histochemical reactions. The problem of fixation at the cellular level is somewhat different from that at the molecular level. At the cellular level where routine histology and cytology operate, it is not necessary to preserve chemical relationships with the same degree of fidelity required for electron microscope studies. With the latter, molecular relationships can be studied and should be preserved, but there are general similarities in the goals desired of fixation, whatever the level that is considered useful. In the living cells, protein, carbohydrate, lipid, and complexes of several or all of these are loosely and weakly associated by hydrogen bonds and salt linkages. These weak associations should be replaced during ideal fixation by stable linkages that will not break down in dehydration, when water is lost from the system. In other terms, soluble substances should be rendered insoluble in those fluids to which tissues are exposed subsequent to fixation. OsmIUm tetroxide (p. HI) is a good fixative for electron microscopy because it reacts with and adds onto many cellular structures, promoting contrast and in a sense "staining" the tissues. Osmium tetroxide is less useful for light microscopy in histology and cytology since most structures are harder to stain after osmium fixation. Probably the usual staining sites are occupied by osmium. On the other hand, some cellular structures stain well after osmium but only when other fixative ingredients are added to the osmium. It is necessary and useful to realize that the universal fixative is not 8

16 Tissue Fixation and Commonly Used Fixatives 9 yet available. Fixation can be controlled to the extent that certain cellular and tissue structures can be demonstrated consistently. We should n.ot delude ourselves that we are working with the faithful images of living cells. The loose chemical and physical associations characterizing life at the molecular level are made abnormally stable and fixed fa: the needs of traditional histologic technique. Tissue fixation is designed to capture rapidly a static picture that retains the closest possible resemblance of the fixed tissue cells to the living cells. The tissue is "killed" in a sense even though, by appropriate methods, many enzymes can be preserved. When tissue is dropped into the ideal fixative, all vital processes stop-especially those of autolysis. Also, basic tissue detail should be so preserved that the subsequent handling (usually dehydrating, embedding, sectioning, and staining) will not detract from its final appearance under the microscope. Place tissue to be fixed in the fixing solution as soon as possible after its removal from the living or dead body. For special purposes, perfusion of the animal or the organ by a fixing solution is feasible. Such perfusionfixation is often preceded by a solution of nitrites or other chemicals intended to empty and dilate the vessels Consult more specialized works for details. The portion of tissue put into a fixing solution should be small enough to allow early diffusion of the fixative into the innermost part of the tissue. Also, the total volume of the fixing solution should be ten to twenty times that of the tissue. Most fixatives harden tissue. As sections were cut freehand in older times, many of the solutions we now call fixatives were originally called "hardening agents." The chemical bases for fixation are still poorly understood. The data have been summarized by Baker (1951). The principles have been discussed by Engstrom and Finean (1958) and Baker (1958). In our own view, it is almost certain that most useful fixatives. produce some degree of polymerization of tissue compounds. This could explain why many tissue materials are rendered much less soluble. This can hardly be due entirely to the denaturation of proteins. In the case of fats and lipid complexes, it is likely that double-bond cross linkages and cross saturation of compounds are :important in fixation. Often, there is some resemblance to the polymerization that occurs,when paint "dries." FUNDAMENTAL TYPES OF FIXATIVES Fixatives are used only as solutions because of the ability of solutions to diffuse into the cells and tissues. They can be classified in several different fashions: according to their effect on proteins, as either precipitating or nonprecipitating, or according to their ability to fix some particular cellular structure, as, for example, fixatives for mitochondria

17 10 Examination of Tissues-Preparing Tissue Sections and Smears and fixatives for the Golgi element. However, none of these classifications is particularly valuable for practical purposes. It is useful to know something about the action of the simple fixatives, which are solutions of just one substance (usually WIth water), and the more usual fixing mixtures, in which several of these compounds are put togejlrer in a more useful form. ~xamples of simple fixatives are alcohol, formaldehyde, acetic acid, picnc acid, chromic acid, potassium dichromate, mercuric chloride, cadmium chloride, cobalt nitrate, osmium tetroxide or osmic acid, and acetone. Fixing mixtures are usually made up of combinations of these simple fixatives and are usually denoted by the name of the person who originated the mixture. For example, Zenker's fluid contains mercuric chloride and potassium dichromate in water. This is Zenker's stock solution. Usually glacial acetic acid is added just before use. Zenker's stock with Formalin added instead of acetic acid is called HeIly's fluid. DaFano and Aoyama produced fixing mixtures for the Golgi element. Altmann and Flemming described osmium mixtures that are particularly valuable for the study of mitochondria and the Golgi element. Each of the Simple fixatives will be described briefly and some of the fixing mixtures will be descrihed The data in these areas are largely taken from Baker, 1958, to which reference should be made for more complete discussions. Basic Ingredients of Fixatives Sometimes Used Singly (Simple Fixatives) Ethyl alcohol (C 2 H s OH) is a colorless liquid, readily mixed with water. It is used in concentrations from 70 per cent to absolute. As a primary fixative alcohol is useful for the preservation of certain enzymes, e g., alkaline phosphatase, but will, of course, dissolve the triglyceride fat of cells and tissue. Proteins are precipitated by denaturation. Alcohol, particularly in the absolute strength, was much used as a fixative for glycogen. It shrinks tissues and hardens them considerably unless used at deep-freeze temperatures. Formaldehyde (H, CHO) is a gas which, in the form that reaches the laboratory, has been dissolved in water at 39 per cent or 40 per cent strength. Thls solution is called Formalin or commercial Formalin. Formalin when diluted (commonly 10 per cent), is the ~ost frequently used fixing solution and is probably the one most worthwhile. It forms additive compounds with proteins and apparently can produce bridging and polymerization. Since Formalin is the aqueous solution of formaldehyde, it does not dissolve out the fats and it appears to be a good fixative for the complex lipids. Formaldehyde tends to break down to formic acid and for optimum use the solution should be buffered. Tissue does

18 Tissue Fixation and Commonly Used Fixatives 11 not shrink very much during Formalm fixation. Anyone who has worked around a laboratory realizes that Formalin hardens tissue, this frequently being most noticeable on the fingers of the laboratory technician. Dilute acetic acid (CHsCOOH) is one of the oldest fixatives, since as the principal active constituent of vinegar it has been used in pickling since ancient times. Baker suggests a range of 0.3 per cent to 5 per cent strength as useful m tissue fixation. DIlute acetic acid penetrates rapidly, does -not affect fats, and precipitates nucleoproteins. Despite the fact that it damages or destroys mitochondria, it is useful because of its good fixation of nuclei. Picric acid (C 6 H2 (N02) soh) is a yellow, crystallme solid, which becomes nearly white when water of crystallization IS removed. Wlule this paler form is more desirable for fixation, it is unfortunately mflammable and can explode. Picric acid is a protein precipitant, forming compounds known as picrates. Picric acid solutions, especially when prepared in alcohol, are useful in the fixation of carbohydrates, e.g., Bouin's fluid. As picric acid has no established primary effect on carbohydrates, its value as a carbohydrate fixative may depend on ItS action on the proteins often associated WIth tissue carbohydrates. The tissue is left yellbw-colored. This color does not interfere with microscopy as it is usually washed away during and after staining. Chromic acid (H2Cr204) in aqueous solutions is more properly called chromium trioxide. It is readily soluble in water and precipitates proteins. It is seldom used in primary fixation except in mixtures, since chromic acid alone has a violent and disruptive action when coming into direct contact with cells. Chromic acid is used also as a post chroming agent, although potassium dichromate mixtures, to be discussed next, are more commonly used for this purpose. Potassium dichromate (K2Cr20 7 ), a water-soluble salt, is an oxidizer and particularly valuable in mixtures for the fixation of lipids, especially phospholipids. In an acid solution it precipitates protein. It appears to dissolve chromatin. Treatment of tissues m 2 per cent to 3 per cent potassium dichromate for 3 to 7 days after initial fixation in Formalin allows much better preservation of myelin and other phospholipids in paraffin sections than is obtained with Formalin alone. Mercuric chloride (HgCI2) (bichloride of mercury) is a precipitant of all proteins and, therefore, finds its use in many fixing mixtures. Left in the tissue is a mercury-containing precipitate that must be removed with an iodme mixture (p. 20). Mercuric chloride IS one of the fixing substances that truly facilitates staining, promoting more brilliant coloration hy most dyes in sections of tissues so fixed. Cadmium chloride (CdCI2) and cobalt nitrate (CoNO a ) are metallic compounds whose use in tissue fixation is restricted at present but may increase when more is known of their mechanisms of action. Such

19 12 Examination of Tissues-Preparing Tissue Sections and Smears metallic salts were introduced by Ramon y Cajal, who found that silver impregnation of the Golgi element was possible after certain fixative mixtures containing salts of uramum, lead, or manganese. DaFano introduced cobalt nitrate for the same process and Aoyama introduced cadmium chloride. These materials appear to form complexes with the phospholipids and other lipids of the Golgi element. This effect can be shown even after primary fixation in conventional fixatives if blocks are then placed in silver solutions for appropriate times prior to dehydration, clearing, and paraffin infiltration. Cadmium and cobalt make useful fixatives for proteins as well and can be used to preserve cellular structure in superior fashion. ' Osmium tetroxide (OS04) is commonly known as osmic acid solution. It is a strong oxidizer and fixes tissues in the best fashion possible in the sense of preserving in the fixed section a more lifelike appearance of the tissue. It vaporizes readily, forming a substance very irritating to the eyes, and can produce severe discomfort and conjunctivitis if not handled with great caution. Osmium tetroxide is soluble in fats and lipids. and fixes them readily while blackening them. It forms chemical complexes indicative of considerable chemical change in the tissues, particularly noticeable if oxidative reactions, e.g., the penodic acid-schiff reaction, are subsequently performed (p. 126). It has the disadvantage of making the tissue very friable and crumbly. As osmium tetroxide penetrates tissues very poorly, blocks to be fixed should be small and thin. Acetone (CHsCO) is a powerful no~polarizing solvent for lipoids that mixes readily with water in practically all strengths. Acetone is much used instead of alcohol for dehydration. Cold absolute acetone ( _5 C.) is useful as a tissue fixative that partially preserves certain enzymes largely or completely destroyed by traditional fixatives. Examples are. lipase and acid phosphatase. Fixative Mixtures No single chemical agent possesses all the attributes desired of a general purpose fixative. Also, many such "simple fixatives" prove to have one or more undesirable effects on tissues that complicate or limit their use. Hence various mixtures of two or more chemicals have been tested in the hope of obtaining composite or additive fixative action superior to that obtainable when anyone of the ingredients is used alone. While some fixative mixtures have been carefully designed for specific purposes, the discovery of other useful mixtures has resulted from trial and error. Countless mixtures have been tried. While the number of fixative mixtures that have proved of decisive benefit is relatively few, the con- \ tributions of these to improved microscopy of tissues have been sub-

20 Tissue Fixation and Commonly Used Fixatives 13 stantial. Nearly all fastidious microscopists tend to use one or more fixative mixtures for general-purpose fixation. Even when Formalin is used routinely, it has become traditional to add alkalinizing agents in order to prevent the acidity present when formaldehyde is merely diluted with-water. The use of fixative mixtures is now well entrenched but has certain disadvantages. Some fixative mixtures proposed contain so many ingredients that analysis of their action in terms of specific effects on particular SUbstances of cells and tissues is most complex if not impossible. While the concept of fixative mixtures has proved valuable, it is difficult to justify the rational use of mixtures that contain a great number of ingredients. The less complicated mixtures should be used wherever possible, in preference to the more elaborate concoctions formerly in vogue. Other considerations, such as the care and promptness with which sufficiently thin blocks of tissue are placed into an adequate amount of fixative, are often just as critical as the particular choice of fixative in determining the final appearance of the microsection. The composition and directions for use of recommended fixative mixtures is discussed later (p. 19). PREPARATION OF THE TISSUE SPECIMEN FOR FIXATION Tissues removed from surgical specimens or the dead body for microscopic study can be damaged and/or adequate fixation prevented by: ( 1) autolysis; (2) pressure; (~) osmotic injury; (4) drying effects; (5) excessive thickness of the slice; and (6) excessive blood and mucus. Autolysis Tissue slices destined for microscopy should be removed from the gross specimen as soon as possible. As this cannot always be done immediately, surgical specimens requiring dissection should be refrigerated promptly and never left for hours at room temperature. Specimens that are sufficiently small are often placed directly into fixative at the time of surgery. Cadavers not autopsied promptly should be placed as soon as possible into a morgue of adequate COOllIig capacity. Actually, it will take many hours before an adult body becomes sufficiently chilled to retard Significantly the autolytic changes that impair microscopic details of cells and tissues. But the prompt refrigeration of the smaller bodies of infants and of. laboratory animals proves even more rewarding in the prevention of such damage; such smaller bodies can be effectively cooled in hours. Ideally, tissues of experimental animals should be removed immediately after death, this is easier when animals can be killed than when death occurs spontaneously. Some workers teach their assistants to

21 14 Examination of Tissues-Preparing Tissue Sections and Smears remove the viscera of animals dying at inconvenient hours and to place the organs en masse into chilled fixative. While the urgency for prompt fixation of the definitive histologic specimens will vary with the particular needs of the microscopist, any delay detracts from the excellence of the final microscopic appearance. Pressure The slice of tissue excised for fixation should be cut with a very sharp knife or razor. Do not allow the delicate structures of cells and tissues to be crushed or excessively distorted by the careless use of forceps or excessive pressure from a dull cutting edge. Scissors cause more crushing along the edges of a tissue block than a knife. Drawing the sharp edge of a long knife back and forth across the gross specimen allows slices to be cut wlth only the slightest pressure being applied directly to the tissue. It is often useful to moisten the knife's edge with physiologic saline solution; this minimizes the tendency of the tissue to stick to the -knife. When forceps or tweezers are used, take care to grasp the tissue as gently as possible by means of the connective tissue, away from the areas of main interest to the microscopist. Tissue slices can be transferred directly from the knife to fixative by immersion or gently dislodged by shaking or wiping. Take care not to damage the knife's edge by contact with certain fixatives capable of corroding metal, e.g, Zenker's fluid. The basic principle here is to excise, trim, and transport the tissue slice to fixative with a minimum of pressure injury to microscopic structure. I Osmotic Injury The common practice of washing tissues wlth water is convenient but causes tissues and cells to swell as some of the water is imbibed by the salt-rich tissues. Such artifacts can be prevented if rinse water of about the same salt concenti'ation as tissue juice is used instead. Red blood cells are also laked by exposure to water but not by osmotlcally correct solutions of saline. Large containers filled with so-called physiologic saline, 0.85 per cent to 0.90 per cent sodium chloride, allow convenient rinsing of specimens without producing swelling of the tissues or laking of blood. ' Drying Exposure of the gross specimen or cut slices of tissue to air for any considerable period causes shrinkage as a result of drying out. Specimens awaiting dissection should be kept moist, preferably with physiologic

22 Tissue Fixation and Commonly Used Fixatives 15 saline; this prevents desiccation by means of a moistening agent causing no osmotic injury in itself. Inexpensive plastic sheeting or bags now available allow surgical specimens awaitmg examination to be stored in the refrigerator WIth less danger of drying out than the former cus~ tom of wrapping them in gauze, all too often dry. Excessive Thickness of the Tissue Slice Before autolytic processes, presumably the result of enzymes acting on devitalized tissue, can be stopped there must be contact with the fixative. If the tissue slice is too thick, autolysis may become advanced inside the tissue before adequate penetration of fixative has occurred. For this reason thinner blocks are fixed better and more rapidly than very thick ones. For ordinary pathologic purposes, tlssue slices should be no thicker than 3 to 4 mm. For more fastldious preservation of finer cytologic details, tissue slices should be no greater than 2 mm. m thickness; thinner slices may prove more difficult to section regularly on the microtome. Any unevenness of very thin slices that may result from curling or imperfect flattening during embeddmg makes it harder to obtain full sections of the original block; such also leaves only a scant margin of safety during shaving or facing of the paraffin block or during recutting of additional sections that may be needed. While the length and width of the slice make little difference in the rate of fixative penetration, slices that are larger than an ordinary postage stamp prove inconvenient to handle in later stages of processing. Excessive Blood and Mucus These tend to coagulate rapidly on the surface of tissues, whether exposed to air or in fixative solution; they may produce a tough coating or film that is penetrated only slowly by fixative. It is better to protect tissues against drying; wash off excess blood or mucus with physiologic saline before tlssue slices are placed in fixative. ADVICE ON THE USE OF FIXATIVES Assuming that tissue slices have been promptly and-properly prepared for fixation, use of the fixative itself requites thoughtful consideration. For handling tissues in fixative, short bottles of clear glass with wide mouths and adequate capacity are recommended. Cork stoppers are convenient and less often damag~d than rubber stoppers by such solvents as acetone. For autopsies or whenever large numbers of slices from a single case are collected, glass-topped Mason jars are useful as fixing bottles. The bottles or other fixatlve can tamers should be cleaned before each use and always adequately labeled. Of the several methods used for

23 , 16 Examination of Tissues-Preparing Tissue Sections and Smears tagging bottles, the safest is to write with pencil on a strip of plain white paper that is then sealed inside the jar and cannot be lost. Other means for taggmg bottles include labels of adhesive tape applied to the out SIde or a card whose attached strmg can be sealed inside the bottle by the mserted stopper. In laboratories usmg automatic tissue-processing equipment, each tissue slice can be placed into a small perforated contamer that, in turn, is placed into a large receptacle, e.g., beaker, containing fixing Huid. Ordinarily, tiny strips of paper wlth identifying data clearly written in pencil or India ink can be placed mside the perforated container along with the tissue. Containers with fixative should be prepared ahead of time so that no delay in tissue fixation occurs while obtaining bottles of fixative. For those fixing mixtures whose ingredients cannot be mixed untu ready for use, weighed or measured volumes of the components can be put into separate bottles that need only to be combined at the time of use. Fixatives should be used at a low temperature, preferably in the refrigerator (5 C.). Whenever fixation at low temperature is planned,. containers with fixative can be stored in the refrigerator until time of use, and promptly returned once the tissue has been collected. This IS a useful practice for ensuring optimal preservation of surgical specimens that are sufficiently small to be placed directly in fixative at the time of surgery. The volume of fixative should be about ten to twenty times the volume of the actual tissue slices. While a somewhat smaller excess of fixative over tissue may cause no obvious harm, instances of tissue spoilage can occur when the amount of fixative is grossly inadequate. The tissue slices should be kept Hat as possible in the fixative. Tissues such as lung that may Hoat can be kept Immersed by placing porous paper or gauze sufficient to cover the tissue inside the fixative container.. Occasional agitation of the bottle containing the tissue and fixative minimizes poor penetration of the latter owing to packing or adherence of slices to one another. Tissues that tend to curl, e g., intestine or skin, can be placed atop a strip of porous paper towelmg or filter paper before immersion in fixative. The less important surface, e.g., serosa or dermis of the examples given, should be placed next to the paper. The paper is naturally removed at some later stage in processing, after the tissue has become stiffened and will remain Hat. A mat of cotton or gauze placed on the bottom of the fixative container beneath the tissue' slices will allow the fixative to enter the tissues from all sides. For special purposes, always consider ways of allowing the preserved tissue slice or sample to be fixed in a more lifelike fashion. For example, lungs with attached bronchi or trachea can be filled with fixing Huid m jected under gentle pressure; the trachea or bronchus can later be clamped to prevent leakage. The entire specimen is then put into a large

24 Tissue Fixation and Commonly Used Fixatives 17 container filled with the same fixing fluid. There are other examples. Saline can be used to wash out a segment of intestine or other tubular structure that is then tied off at one end and filled with fixative. The other end is then tied or clamped and the, specimen placed into a container of similar fixative. Smaller blocks for histologic processing are r~moved from such fixed gross specimens after 12 to 24 hours of preliminary fixation. More elaborate attempts to obtain more faithful fixation include the fixing of an entire aorta filled with fixative under pressure corresponding to systolic or other level of blood pressure desired. The aorta is also immersed in fixative while the fixative in the lumen is kept under pressure by tying off all branches except its connection to the mercury pump, which is checked at intervals. Such treatment for only five to six hours produces sufficient stiffening that the more lifelike shape of the wall is preserved in the annular segments that are cut, the fixation of which is completed in ordinary fixing bottles. Very tiny biopsy specimens are sometimes lost unless great care is taken to wrap them in porous paper, e.g., empty tea bags or tissue paper, or in gauze. As the importance of such tiny specimens may be great in spite of their size and the difficulties they present, one cannot overemphasize the necessity of scrupulous care to avoid their loss. The duration of fixation is of some importance. As will be detailed later, some fixatives allow for almost indefinite preservation of tissues. As a general rule the optimum fixation is the shortest necessary for the tissue cells. Further tissue handling should proceed as expeditiously as pos SIble. Buffered neutral Formalin (p. 19) will fix a block of tissue 1 X 2 X 0.4 cm. in 4 to 6 hours. Finally, the best time for fixation of pathologic tissues is the moment the specimen or body comes into the hands of the pathologist. This moment is the optimum possible, and the last opportunity for optimal fixation. It may be posslble to freeze some of the tissue and to fix it subsequently but this is a poor substitute, as a rule, for primary fixation. GENERAL PURPOSE FIXATIVES A well-equipped laboratory for the study of tissues by the available modem methods should have at least these three groups of fixation solutions always at hand: - \ per cent neutral buffered Formalin, this is the most useful general fixative and allows the majority of routine studies as well as a number of special procedures, including histochemical tests. 2. Cytologic fixatives : ReIly's fixative for various cytoplasmic structures and DaFano's for the Golgi element; and 3. Fixatives for specific histochemical purposes, e.g, cold alcohol Formalin for polysaccharides, cold acetone for certain enzymes, etc.

25 . 18 Examination of Tissues-Preparing Tissue Sections and Smears It is preferable to keep all these fixatives at refrigerator temperature before and during use. For sirigle specimens and for surgical specimens it is worthwhile to keep these groups of fixatives prepared and bound together with adhesive tape for convenience of use. In some instances it will be desirable or possible to use only the basic fixative, Formalin-preferably the neutral buffered solution. A number of enzyme studies are possible in frozen sections cut from tissue fixed for several hours in cold neutral buffered Formalin (p. 19). Whenever the cytologic studies that require fixative are possible, histochemical studies are also possible ai~d these can be done on material fixed in the fixatives of Group 3. Ten Per Cent Formalin. Commercial Formalin (approximately 40 per cent solution of formaldehyde gas in water) tends to develop formic acid on standing, causing an acidic reaction with litmus. Such acidic F<;>rmalm when diluted tenfold, i.e., 10 per cent Formalin, has good fixing properties but causes the formation of an objectionable brown. pigment (Formalin pigment) to appear in the final microsections. This pigment results apparently from the interaction of acidic Formalin with hemoglobin, and is usually more abundant in tissues that are rich in blood and in blood vessels. While usually recognizable, such pigment can be mistaken for hemosiderin or malarial pigment; it tends to interfere with the histologic detection of tiny structures such as microbial agents and may cloud other histologic details If especially abundant. The usual 10 per cent solution of Formalin (4 per cent formaldehyde) is made as follows. 10% Formalin I Commercial FormalIn Distilled water 10 mi. 90 ml. Some neutralization of formic acid is usually attempted by layering calcium carbonate, usually in the form of chalk dust or marble chips or magnesium carbonate, at the bottom of a large stock container of diluted Formalin. The degree of antagonism of the acidity or neutralization obtained is variable and inexact. Samples of Formalin withdrawn into fixmg bottles soon become acidic on storage or during use as fixative. While these measures may minimize the production of Formalin pigment, the protection is only partial and not dependable. Lillie's Neutral Buffered Formalin (1948). Much greater constancy and more dependable protection against acidity of Formalin are obtained by use with a dilute phosphate buffer. While a requisite for more precise histochemical work, Lillie's neutral buffered Formalin is recommended for general usage in the histopathologic laboratory. Its use al-

26 Tissue Fixation and Commonly Used Fixatives 19 lows virtual freedom of microsections from formalin pigment. It IS made as follows. Lillie's Neutral Buffered Formalin Commercial Formalin (37%-40% formaldehyde) DIstilled water Sodium acid phosphate, monohydrate (NaH,PO. H,O) Disodium phosphate, anhydrous (Na,HPO.) 100 m!. 900 ml 4.0 Gm. 6.5 Gm. Tissues being fixed at 4 C. can usually be left in the solution up to 72 hours. However, overnight fixation is adequate if the tissue slices are no thicker than 3 mm. Some procedures require only a few hours. A convenient feature of 10 per cent Formalin as a fixative is that tissues can be stored in it indefinitely. Zenker's Fluid. This is a mixture of potassium dichromate and bichloride of mercury in a stable stock solution to which glacial acetic acid is added just before use. TradItionally, most workers use a final concentration of 5 per cent acetic acid but some authonties have preferred 10 per cent acetic acid. Zenker's stock is prepared as follows. Zenker's Stock PotaSSIUm dichromate (K,Cr,O,) MerCUrIC chloride (HgCI,) Distilled water 25 Gm. 50 Gm. 100 m!. Dissolve the two salts in the water with the aid of heat (Older directions prescribed the addition of 1 Gm sodium sulfate to the dissolved dichromate and mercuric chloride, but this is no longer considered of any value and has been discarded.) As the stock solution is stable, It is useful to prepare large quantities for storage until needed. For use, Zenker's fluid is prepared by combining as follows. Zenker's Fluid Zenker's stock Glacial acetic acid 100 m!. 5 m!. Helly's fluid is also prepared from Zenker's stock except that formaldehyde instead of acetic acid is added, just before use. This fixative is also known as Zenker's with formaldehyde or Zenker-formol. Do not confuse Helly's with Zenker's fluid. Helly's fluid is prepared just before use as follows.