I A Simple Technique for Staining of Cell Membranes with Imidazole and Osmium Tetroxide'

Transcription

1 /$3.30 The Journal of Histochemistry and Cytochemistry Copyright by The Histochemical Society, Inc Vol. 43. No. 10. pp Printed in US.A. Technical Note I A Simple Technique for Staining of Cell Membranes with Imidazole and Osmium Tetroxide' GEORGES THIERY, JACQUES BERNIER, and MICHEL BERGERON2 Deparimen i of PbysioLogy, Universiii de MoniriaL, Montriad Qu6&ec, Canada. Received for publication February 8, 1995 and in revised form May 3, 1995; accepted May 25, 1995 (5T3592). We describe a simple new technique based on the affinity of imidazole and osmium tetroxide for unsaturated lipids. Organs (e.g., kidney, liver, intestine) were perfused in vivo with a glutaraldehyde solution. Tissue fragments were then immersed in a solution containing imidazole and Os04 and are further stained with a double lead and copper citrate solution. Ultra-thin (0.06 pm) or thick ( pm) sections were observed with transmission electron microscopy ( kv). The method presented permits excellent visualization of cell membranes (e.g., endoplasmic reticulum, endocytotic apparatus) because it favors good resin penetration and the alkaline ph preserves cell volume. A better stereomicroscopic analysis of the relationship between cell organelles can be carried out with thick sections. The imidazolelosmium can be used routinely because the technical steps are easy and simple to follow. Furthermore, it can complement other cytochemical methods. (J Hisrochem cyfochem 43: , 1995) KEY WORDS: Endoplasmic reticulum; Cytomembranes; Histochemistry; Kidney; Liver; Rat; Imidazole; Osmium tetroxide. Introduction The use of en bloc staining coupled with thick sections ( pm) has provided new views on the spatial organization of cell organelles in the cytoplasm, i.e., mitochondria (I), Golgi apparatus (2,3), and endoplasmic reticulum (4-7). However, metal impregnation has a major drawback because it cannot offer clear images of cell membranes and therefore the relationships among organelles cannot be observed with precision. When post-fixed with potassium ferrocyanide and osmium (8), cell membranes could be visualized in pm sections. However, because the many plasma membrane infoldings act as a barrier to resin penetration, the sections become too fragile to resist the electron beam and are difficult to observe. We present a new method, based on the affinity of imidazole and os04 for unsaturated lipids, that has been already demonstrated by Angermuller and Fahimi (9). This method appears to achieve better resin penetration than osmium/ferrocyanide and therefore produces excellent contrast for membrane staining. Supported by grant MT-2862 from the Medical Research Council of Canada and by the Minisdie de I'Education of the Government of Qutbec, Canada. ' Correspondence to: Dr. Michel Bergeron, Dept. de physiologie, Facultt de Medecine. Universite de Montrtal, CP 6128, Succ. Centre-ville, Montreal, Quebec, Canada, H3C 3J7. Materials and Methods All chemicals used were of premium grade and were purchased from BDH (Toronto, Ontario, Canada). Imidazole was purchased from Baker (Philipsburg, NJ). Glutaraldehyde, 0 ~ 0 4 and, epoxy resin were obtained from Mecalab (Montrtal, Quebec, Canada). Preparation of Sohtions Primary Fixative. One of the four following solutions was used as a primary fixative: a. Glutaraldehyde 2.5%/citrate/barium, ph 8.6, composed of 0.1 M barbital-acetate, ph 8.7 (6 vol), 0.1 M barium chloride (2 vol), 0.1 M trisodium citrate (1 vol), and glutaraldehyde 2.5% (1 vol). b. Glutaraldehyde 2.5%/citrate/calcium (ph 7.2) (5). c. Glutaraldehyde 2.5% in 0.15 M phosphate buffer (ph 7.2). d. Periodatdlysindparaformaldehyde (PLP)(10) alone (ph 6.5) or, for better fixation, fragments were dipped for 45 min at 20'C in a solution of glutaraldehyde 2.5% (Solution b, above) at a final ph of 7.2. Washing Solutions. Barbital-acetate 0.1 M (ph 7.2) or distilled water wz used as washing solution. Post-fixative. The post-fixative solution was made up of 0.1 M barbitalacetate, ph 7.2 (2 vol), 0.1 M imidazole in 0.1 M barbital-acetate (1 vol; ph corrected at 7.2 with HCI), and 2% os04 (1 vol). Staining solution. The double lead-copper citrate solution described by Thitry and Bergeron (1) was used for staining. A mixture of 1 M lead nitrate (2 ml) and 0.4 M copper sulfate (0.5 ml), followed by 1 M NaOH (4 ml), was slowly added, stirring constantly, to a beaker containing dis- 1079

2 1080 TH~RY, BERNIER, BERGERON tilled water (7 ml) and 0.2 M trisodium citrate (13 ml). The blue solution could be used immediately. Tissues had to be fixed at a perfusion rate that was roughly twice the vascular rate of a given organ. In this study, male Sprague-Dawley rats ( g) were perfused for about 10 sec with Locke or Hanks solution containing heparin sodium (2 mu/100 ml), and for 2 min with one of the primary fixative solutions described above. To perfuse the fixative solution into both kidneys, a clamp was applied below the renal arteries and thus a catheter could be inserted in the abdominal aorta below the renal arteries. The mesenteric artery was ligated, the blood flow from the thoracic aorta was blocked by a ligature above the renal arteries, the aortic clamp was released, and perfusion was immediately started at the rate of 6 ml/min. Twenty to 30 sec after initiation of perfusion, the jugular vein was sectioned. For the intestine, the same procedure was followed except that the ligature was placed above the mesenteric artery, which was not ligated. The perfusion rate was 12 mllmin. For the liver, the catheter was inserted into the portal vein and the perfusion rate was 30 ml/min. The tissues were cut into small fragments 0.5 mm thick and immersed in the same fixative solution for 45 min at room temperature. They were then washed three times for 5 min in the 0.1 M barbital-acetate buffer and incubated in the postfixative solution for 1 hr at 37 C. The fragments were subsequently washed three times for 5 min with distilled water and stained for 30 min at 37'C in the double lead and copper citrate solution. For large fragments ( mm thick), the staining time with Pb/Cu citrate had to be increased to 1 hr. If the vials or their caps were made of polyethylene, specimens had to be transferred to new vials after the imidazole/osmium fixation to prevent the precipitation of the osmium that reacts with polyethylene. Fragments were again washed three times for 5 min with distilled water and epon embedding was carried out in the usual manner with a propylene oxidelepon niixture (1:1 for 2 hr and 1:2 for a minimum of 12 hr). Thick sections of pm were made with a Reichert Ultratome and were observed with a transmission electron microscope (Philips 300) at 80 or 100 kv. Results Thick sections of pm, observed by standard transmission electron microscopy, showed excellent contrast, thus facilitating a tri-dimensional study. The thickness of 0.15 pm appeared to be ideal and proved to be better than ultra-thin sections (0.06 pm). However, organelles appeared to be dense at a thickness greater than 0.3 pm. As shown in Figures 1 and 2, better spatial visualization of each organelle was possible because the various cell membranes were well stained and the cavities of the organelles appeared transparent. Membranes appeared much less granular with imidazole osmium than with the other en bloc staining techniques. Because the nuclei and the ribosomes were unstained, the cytosol appeared transpar- ent and the fine characteristics of the ER membranes could be easily detected. However, the distinction between RER and SER was not easy to make because of the absence of ribosomes. Staining of the various cell membranes was more or less uniform, with the exception of the membranes and cristae of the mitochondria, which were more intensely stained. The GZJ saccules of the Golgi apparatus were more readily visible in cross-sections (Figures 1 and 2). as expected. This method of staining also revealed different types of dense bodies which, according to the observations of Angermuller and Fahimi (9), corresponded to very low-density lipoproteins (VLDLs). When the fixation was carried out after perfusion of the vascular system with a physiological solution (Locke, Hanks), dark-colored oblong bodies or filamentous masses could be seen in the intercellular spaces or in the dense apical tubule systems of the proximal nephron. They most likely also represent VLDLs (Figures 2 and 3) and were also observed in the Golgi saccules of hepatocytes (Figure 5). All four of the above-mentioned fixative solutions gave satisfactory results. To preserve antigenicity, weak fixation with PLP is generally used for immunofluorescence studies; subsequent addition of glutaraldehyde/calcium citrate for 45 min is therefore recommended for better contrast or for optimal preservation of structure. Similarly, better results were obtained with the fixative solution at ph 8.6 (Solution a) than at ph 7.2. The presence of trisodium citrate is essential in osmic impregnation (Figure 4), but it does not appear to be essential in the imidazole/osmium technique and can therefore be omitted. We have generally used a fixative containing trisodium citrate to compare the results obtained with imidazole/osmium and with the osmium impregnation. The ER organization and characteristics were similar with both methods, despite the fact that the imidazole method stains membranes and the osmium impregnation stains the cisternal content. Worthy of note, the canaliculi and especially the fenestrated saccules of the ER network were better delineated with the imidazole technique than with the metal impregnation (compare Figures 2 and 4). The endocytotic apparatus of the apical tubule cells was clearly stained with imidazole (Figure 3), a characteristic not seen with the heavy black osmium deposits. Discussion Imidazole-buffered tetroxide was introduced by Angermuller and Fahimi (9) to visualize lipids by transmission electron microscopy. However, their method had the disadvantage of forming precipitates with Os04 in the solution at high concentrations. Given that Figure 1. Tubule cell of the S3 segment of the proximal nephron. Note the fenestrated saccules (arrowhead) lying against the intercellular membrane (arrow) and surrounding many mitochondria (double arrow) and lysosomes (L). Elements of the Golgi apparatus are seen in cross-section (asterisk) and also tangentially (double arrowhead). Fixation by perfusion with 2.5% glutaraldehyde in barbital buffer, citrate-calcium, post-fixation with imidazole/osmium and lead/copper citrate stain. Original magnification x 29,000. Bar = 0.5 pm. Figure 2. Tubule cell of the S3 segment of a proximal nephron. The ER network is made of intricate tubules recalling the shape of a sponge@). This type of ER network appears to differ in morphology from the fenestrated saccules (free arrowhead) or that surrounding the mitochondria and the lysosomes. Small vesicles can be seen on the cis face (c) of the Golgi apparatus; vesicles on the trans face (t) are larger than on the cis face. One multivesicular body (V) lies near the Golgi apparatus (asterisk). A few dense bodies are present in the intercellular spaces (arrow) and are most likely VLDL lipoprotein. (Inset) Higher magnification of the Golgi apparatus showing the trans phase arranged as a crescent on the left side and the cis phase on the right side near the nucleus. Fixation by perfusion with 2.5% glutaraldehyde in barbital buffer, citrate-calcium, post-fixation with imidazolelosmium and leadkopper citrate stain. Original magnification x 30,000; inset x 45,000. Bar = 0.5 sm; inset = 0.2 pm. +

3 1081 CELL MEMBRANE STAINING WITH IMIDAZOLE-OSMIUM TETROXIDE ~~ ~ ~ ~ -_--

4 THICRY. BERNIER. BERGERON / r n, I,

5 CELL MEMBRANE STAINING WITH IMIDAZOLEOSMIUM TETROXIDE 1083 only low concentrations of imidazole are required in this new method, the solution remained clear and therefore diffused rapidly, especially at 37 C. Tissue fragments were thoroughly washed, because otherwise the phosphate groups would form precipitates with lead citrate, producing a slight but visible background on the photographs. Moreover, it must be emphasized that although the use of lead salt was not recommended for staining ultra-thin sections exposed to imidazole (11,12) because of the possibility that lipids would be extracted, the use of the double PblCu citrate SOlution for staining en bloc did not appear to modify the integrity of the membrane lipids. In particular, some lipid structures (most likely VLDL bodies) were perfectly visible in the Golgi apparatus of the hepatic cells (Figure 5). Interestingly, this technique yielded satisfactory images without the use of uranyl and/or lead salts for ultra-thin sections. The ph of the perfusion solution was a key factor and may be important in certain studies, because acid fixatives appeared to induce cell contraction, whereas an alkaline ph appeared to preserve not only the initial cell volume (13) but also the membrane interrelationships of the various organelles. This certainly constitutes another advantage of this newly described technique. The imidazole/osmium technique, which is easy to apply, can also be used to complement immunofluorescence studies of sections briefly fixed with PLP. However, when control samples were observed with standard transmission electron microscopy, it was necessary to complete the weak PLP fixation with glutaraldehyde. In comparing the ER organization in sections stained by metal impregnation or imidazole/osmium, the same ER network images were observed (Figures 2 and 4). With imidazole/osmium, all cells were consistently stained, whereas with osmic impregnation no reaction took place under the same normal conditions in some cells adjacent to others that were well impregnated. This phenomenon was observed in various tissues (e.g., proximal nephron, prostatic secretory cells, jejunum columnar cells, rat stem cells, toad bladder cells, flounder gut cells) and seemed to be associated with a histochemical property of the ER cisternae content relating to metabolic or hormonal cellular effects, rather than to a structural modification (5-7,14-18). The fact that the ER of all cells is stained with imidazole/osmium confirms this interpretation and further illustrates that the osmium impregnation technique constitutes a histochemical staining, as suggested earlier. Acknowledgments We gratefully acknowledge the asdance of MJ Christiane Laurier for secretarial work. Literature Cited 1. Thitry G, Bergeron M. Morphologie spatiale des mitochondries des tubes proximaux et distaux du nephron. Rev Can Biol : Friend DS, Murray MJ. Osmium impregnation of the Golgi apparatus. Am J Anat 1965;117: Rambourg A, Chrttien M. L appareil de Golgi: examen en microscopie electronique de coupes epaisses (0,s-1 p) aprk impregnation des tissus par le tetroxyde d osmium. CRAS Paris 1970; Thitry G. Colorations signaletiques electives sur coupes ipaisses du rtticulum endoplasmique, de la chromatine et des surfaces cellulaires libres des cellules animales. Biol Cell 1979;35: Bergeron M, ThiCry G. Three-dimensional characteristics of the endoplasmic reticulum of rat renal tubule cells, an electronmicroscopy study in thick sections. Biol Cell 1981:42:43 6. Thiery G, Gaffiero P, Bergeron M. Three-dimensional characteristics of the endoplasmic reticulum and the columnar cell of the rat small intestine. An electron microscopy study in thick section. Am J Anat 1983;167: Danechi K, Bergeron M. Difftrence entre les rtticulums endoplasmiques de deux types de cellules sensibles 1 la vasopressine. Mtdecine Sciences 1989;5: De Bruijn WC. A modified os04 (double) fixation procedure which selectively contrasts glycogen. In Bocciarelli DS, ed. Proc 4th Congres Eur Microsc Electron. Rome: Tipographia Poliglotta Vaticana, 1968: Angermiiller S, Fahimi HD. Imidazole-buffered osmium tetroxide: an excellent stain for visualization of lipids in transmission electron microscopy. Histochem J 1982;14: MacLean IW, Nakane PK. Periodate-lysine-paraformaldehyde fixative. A new fixation for immunoelectton microscopy. J Histochem Cytochem 1974;22: Neiss WF. Extraction of osmium-containing lipids by section staining for TEM. Histochemistry 1983;79: Nickerson PA. Lipid droplets in the adrenal cortex of the rat. Preservation after tannic acid-paraformaldehyde-glutaraldehyde fixation and extraction during staining. Tissue Cell 1983;15: Thitry G. Les fixations acides en microscopie Electronique. Proc 7th Congrss Intern Microsc Electron, Grenoble 1970;1:393 Figure 3. Slightly oblique section through the apex of a proximal tubule cell (S2 segment) below the brush border (BB). Many endocytotic vesicles (E) and elongated tubules (arrowhead) appear to be in communication. Some VLDL dense bodies are seen in the vesicles or in the endocytic tubules (double arrow). Fixation by perfusion with 2.5% glutaraldehyde in barbital buffer, citrate-calcium, and post-fixed with imidazole/osmium and leadlcopper citrate stain. Original magnification x 27,000. Bar = 0.5 vm. Figure 4. Osmium impregnation of the same kidney, showing an identical morphology as in 53 tubules. The ER is very well impregnated. The intercellular space is somewhat enlarged by the cell retraction produced by the fixation technique (arrow). Fenestrated saccules surround mitochondria (double arrow); a portion of the paramembranous cisternal system is seen along the lateral cell membrane (arrowhead). The pores of the perinuclear saccule are well delineated. N, nucleus; m, mitochondrion. Fixation by immersion with 2.5% glutaraldehyde in barbital buffer, citratetalcium. Original magnification x Bar = 2 vm. Figure 5. Section through a hepatocyte at the level of the Golgi apparatus (asterisk). Note clusters of VLDL in the trans network of the Golgi (arrowhead). The ER is formed of canaliculi (arrow) and fenestrated saccules (double arrow). m, mitochondrion: G, site of glycogen clusters. Fixation by perfusion with 25% glutaratdehyde in barbital buffer, citrate-barium at ph 8.6. Original magnification x 31,000. Bar = 0.5 pm.

6 LO84 TH~RY, BERNIER, BERGERON 14. Gaffiero P, Bergeron M, Thiiry G. Morphological study of cell organelles during development. I -The nuclear sac and the endoplasmic reticulum of the rat nephron. Biol Cell 1983;49: Beaudry-Lonergan M, Thiiry G, Bergeron M. Osmium impregnation of the endoplasmic reticulum correlates with the function status of prostatic secretory cells. Biol Cell 1985;54: Berthelet F, Beaudry-Lonergan M, Bergeron M. Proliferation of the endoplasmic reticulum of the proximal nephron cell during chronic metabolic acidosis and after treatment with triamcynolone. In Guder W, Kovacevic 2, eds. Biochemical aspects of kidney functions. Berlin, New York: Walter de Gruyter, 1987: McLeese J, Bergeron M. Fasting induces modifications of the endoplasmic reticulum in intestinal cell. J Electron Microsc Tech 1990;16: Danechi K, Hoang T, Bergeron M. Reversible histochemical modifications of the endoplasmic reticulum following AVP stimulation of toad bladder granular cells. Cell Tissue Res 1995;280:365