Procedure 2 Plastic Removal

Tissue Fixation and Embedding in Plastic

1. Dissect tissue to 1-mm cubes or smaller and fix for 2 h at room temperature.

2. Wash tissue 2 x 5 min with PBS.

3. Quench free aldehyde groups with 0.1 M glycine/PBS, 2 x 10 min.

5. Dehydrate with 35%, 50%, and 70% EtOH, 10 min each, on shaker.

6. En bloc stain with 3% uranyl acetate/70% EtOH, 1 h.

7. Continue dehydration through 90%, 95%, and 100% ethanol, 10 min each, on shaker.

8. Prepare epoxy resin and use in 1/1 mixture EPON/ethanol. Infiltrate tissue for 1 h on shaker with EPON/ethanol in capped vial.

9. Prepare 2/1 mixture of EPON/ethanol and infiltrate tissue for 1 h on shaker with cap removed.

10. Prepare fresh epoxy resin, infiltrate tissue on shaker 1 h in uncapped vial.

11. Change epoxy and infiltrate overnight under vacuum.

12. Prepare fresh epoxy resin and embed tissue in Beem capsules or coffin molds.

13. Polymerize blocks in vacuum oven at 55°C for 48 h.

Sectioning, Plastic Removal, Immunostaining, and Re-Embedding Thin Sections

1. Prepare grids for collecting sectioned material: Acetic acid wash nickel grids in a water bath sonicator, rinse with dd-H2O three times in the sonicator, and finally rinse with absolute EtOh and air dry. Prepare formvar film by stripping 0.25% formvar off a precleaned glass microscope slide onto a cleaned dd-H2O surface. Place the grids, matte side down, onto to the film and pick up formvar with grids attached with a sheet of parafilm. Allow film to air dry protected from dust. Carefully remove individual grids from the parafilm with forceps, and place, formvar side down, onto a clean microscope slide. Evaporate a medium coat of carbon onto this "reverse" side of the grids (see Notes 49 and 50).

2. Collect 80- to 100-nm sections on the formvar side of the prepared grids and air dry (see Note 51).

3. Prepare a 1:3 dilution of saturated Na ethoxide with absolute ethanol. When not in use, keep this solution covered to retard evaporation and surface NaOH crystal formation.

4. Immerse individual grids, using anticapillary forceps, in the removal solution for 2-5 min (see Note 52). Wash with three consecutive 1 min immersions in absolute EtOH and hydrate through a graded alcohol series, 1 min each, to filtered dd-H2O.

5. The "reverse" carbon side is blotted with filter paper and the grid is floated on an individual droplet of filtered 50 mM TBS on parafilm, where it remains as subsequent grids are prepared for immunostaining (see Note 53).

6. Using a humidified chamber for all incubations, transfer grids from TBS to individual filtered blocking buffer droplets for 10 min, and wash by floating grids on three consecutive droplets of diluent/washing buffer, 1 min each.

7. Transfer grids to droplets of primary antibody diluted in filtered diluent (see Note 54).

8. Incubate overnight at 4°C in a humidified chamber.

9. Wash the grids by floating on six successive drops of filtered wash buffer over 15 min.

10. Incubate on colloidal gold-conjugated secondary antibody diluted 1/50 in filtered diluent 1 h in the humidified chamber at room temperature.

11. Wash the grids by floating on eight successive drops of filtered wash buffer over 15 min, followed by three successive drops of filtered PBS.

12. Fix the antigen/antibody complex with 2% glutaraldehyde in PBS for 10 min and rinse on 4 drops of PBS.

13. Postfix the tissue with 1% OsO4 in PBS for 10 min and wash on 3 droplets of filtered dd-H2O.

14. Proceed to reembedding the thin sections by immersing each forceps-held grid through the graded alcohol series, 1 min each, and in absolute EtOH 3 x 1 min.

15. Immerse in 2% EPON in absolute ethanol 2 min and carefully blot between two pieces of No. 50 Whatman filter paper.

16. Position grids upright in razor blade slits in the back of a silicon rubber embedding mold and polymerize overnight to 48 h at 60°C.

17. Post-stain with 2% aqueous UA and lead citrate.

18. Examine in the transmission electron microscope.

4. Notes

1. Surfactants such as detergents, oil vapors, and grease from fingerprints interfere with formation and continuity of protein monolayers.

2. Something to consider in choosing sample buffer conditions is that, in the final cytochrome C/sample solution, at pH 9 and above, basic residues on cytochrome C become neutralized. This may result in less protein coating of the negatively charged molecules. At higher salt concentration (above 0.2 M), electrostatic interactions between polyanions and polycations become significantly reduced, resulting in less cytochrome C binding to the negatively charged molecule.

3. Preparation of collodion support films is probably the most important source of day-to-day variation in quality of the final molecular spread image. The goal is to prepare a hydropho-bic surface to come in contact with the hydrophobic protein monolayer. The light carbon coat is useful in achieving an appropriate collection surface. A light carbon coat has been shown to yield proteoglycan monomers with extended disaccharide side chains, while an uncoated collodion surface yields mononers with side chains condensed onto the protein core. A quick indicator of the efficiency of protein pickup is a very flat meniscus of the collected droplet.

4. Results of the stain combination include the discrimination of various tissue components: coarse and delicate elastin fibers are black, collagens appear bright yellow, sulfated macromolecules (proteoglycans, glycosaminoglycans, and secreted mucins) are blue/ blue-green, nuclei are dull red/black, erythrocytes are bright red, while the myofibrils (contractile apparatus of smooth muscle cells and striated muscle) are red/orange.

5. While tissue fixed in methacarn (acid/alcohol) can be used, it results in less satisfactory staining.

6. Prepared biotinylated probe is stored unaliquoted at approx 100 ^g/mL dissolved in a 50/50 (v/v) mixture of glycerol and 0.15 M NaCl at -20°C. This stock preparation does not lose detectable amounts of binding activity when stored for more than a year in this manner.

7. We achieve the best results when cover slips are incubated face up on the parafilm and the solutions are applied directly to the cells. We do not recommend putting parafilm squares on top of the incubation solutions. Evaporation is negligible if covered with the lid from the culture dish.

8. It is of interest that a radioactive isotope of ruthenium red, 103Ru, has been used to estimate glycosaminoglycans quantitatively after separation by electrophoresis (29).

9. We performed radiolabeling experiments to assess the loss of sulfated proteoglycans from fixed cultures of arterial smooth muscle cells and found that approx 40% of the total radiolabeled proteoglycans were lost during routine processing for electron microscopy (30). Inclusion of ruthenium red in the fixatives reduced the losses to less than 1%!

10. There is no one ideal tissue fixative, the goals being to maintain adequate tissue integrity for interpretation while preserving antigen/antibody recognition. Buffered 4% formaldehyde or methyl carnoys (10 mL acetic acid, 30 mL chloroform, 60 mL methanol) for 2 h, are common first choices. During tissue processing into paraffin, avoid temperatures exceeding 56-58°C.

11. Never allow sections to dry out.

12. Bovine serum albumin should be globulin free (immunohistochemical grade) when used to block nonspecific protein interactions.

13. Solutions A and B may be premixed and stored frozen as stocks. Complete resin should be made fresh immediately before each use by mixing room temperature A and B, adding DMP-30, and mixing thoroughly again. Bubbles can be pumped out in a vacuum chamber

14. This fixation and embedding scheme has proven useful as a standard first choice because the degree of molecular cross-linking by the low glutaraldehyde content results in the retention of recognizable epitopes and reasonable ultrastructural morphology. The low viscosity, tolerance of small amounts of water, and low temperature of polymerization also contribute to its being chosen when no ultrastructural localization of a particular antigen has been previously tried in our lab.

15. Use of methanol in the dehydration scheme is an attempt to reduce the amount of extraction during the dehydration process.

16. Use 50- to 100-^L reagent droplets on parafilm sheets for incubations.

17. Millipore (0.22-^m) filter all diluents, buffer washes, and water.

18. Handle grids with nonmagnetic #3 Dumont forceps on the grid rim only. LR White sections tend to swell during staining because of the resin's hydrophillic nature and become extremely fragile, so that touching them with forceps or filter paper can easily cause folds or damage.

19. All buffers and H2O for section washes are 0.22-^m millipore filtered.

20. One microgram of protein denatures to cover 9 cm2, so the approx 3.7 ^g delivered here should spread easily across the entire surface in front of the slide. One source of excessive graininess and low sample contrast is overcompression of the cytochrome C film.

21. The substrate surface should be as flat as possible, so that it receives a uniform metal coating.

22. Vacuum cleanliness and ultimate bell-jar pressure contribute to the final metal coating quality. If the vacuum is not at least 5 x 10-5 torr during evaporation, the mean free path of evaporated metal will be less than the distance from the filament to the sample. Metal vapor colliding with gases will cool off, slow down, and may aggregate into coarse particles, resulting in an excessively grainy shadow.

23. Slowly heating metal just to the evaporation point results in the finest granularity (because of local vapor pressure characteristics).

24. The Bouin's-fix mordant step improves the color quality of primarily the crocein scarlet-stained components.

25. As staining time is increased, there is a decrease in the differentiation between crocein scarlet (erythrocytes, fibrin) and acid fuchsin (myofibrils, smooth muscle) stained tissue components.

26. The phosphotungstic acid step that removes excess Musto elastin stain is progressive and will, if extended, begin to decolorize the fine elastic fibers.

27. Acetic acid removes excess PTA

28. Limit the duration of the final ethanol rinses, as saffron (collagen) will destain.

29. If extracellular hyaluronan is to be visualized, omit the hyaluronidase pretreatment.

30. In some cells, overfixation (i.e., 4% paraformaldehyde for several hours) can result in unintentional permeabilization and/or higher background staining of mitochondria with the Streptavidin-Texas red.

31. Controls for intracellular staining and specificity include omitting the permeabilization step; digestion with hyaluronidase a second time, following permeabilization; and preincubation of the bHABP with excess hyaluronan.

32. If it is necessary to conserve reagents, cover slips can be cut in half using a diamond pencil to score the coverslip. 100 ^L is the minimum volume that will cover a full 22-mm cover slip without excessive evaporation.

33. Cover slips are preferable to chamber slides if they will be viewed with a 100x oil objective.

34. Although cellular morphology is somewhat compromised, air drying preserves the fragile hyaluronan network. The relatively rigorous critical point drying techniques tend to wash away the hyaluronan filaments, as well as other extracellular matrix components, leaving a very smooth cell surface.

35. The coating may cover some of the hyaluronan filaments, especially farther away from the cell. Thus, coating times may have to be determined empirically.

36. The presence of phosphate ions in buffer vehicles with ruthenium red results in the precipitation of OsO4-RR before the critical OsO4/RR/polyanion reaction takes place.

37. Thaw and bring to room temperature.

38. Ruthenium red penetration is dependent on the histological structure of a particular tissue defining the diffusion pathway into the interior and the progressive concentration change of dye at different depths along this pathway. Extended fixation time allows the binding of ruthenium red to ionizable carboxylic acid groups and subsequent oxidation to ruthenium brown at increasing tissue depths as well as the oxidation of the polysaccharide substrate generating new carboxyl groups for RR binding.

39. Inclusion of RR in the primary fixative and intervening washing buffer is not necessary for the critical reaction but is included to limit the possible leaching away of bindable substances.

40. The greatest contrast of RR staining is apparent in sections with no poststain, but fine detail of other tissue structures is revealed when uranyl acetate and lead citrate are used.

41. Hydrogen peroxide potency must be considered; bubbles on the inside of the bottle indicate adequate activity in 30% solutions.

42. Plan on 50-150 ^L of solution/tissue, depending on the size of the section.

43. Including 0.04% NiCl in the developing reagent changes the color of the reaction product to blue/black.

44. The specific enzymes and digestion time required must be worked out for each primary antibody. Excessive or inadequate digestion will result in an altered epitope or an inadequately unmasked antigen respectively.

45. Sodium azide is an inhibitor of peroxidase activity and should not be included in buffers used to make the peroxidase substrate or the ABC reagent.

46. Tissue may be harvested after perfusion fixation with 3% paraformaldehyde if desired and then continue fixation in 3%/0.25%.

47. Never allow the section surface to dry between droplet changes.

48. Include appropriate buffer/no-primary, irrelevant antibody, and positive and negative tissue controls.

49. Extreme care should be used in handling coated grids, as an intact support film limits difficulties later with contamination and section deformation. The use of clean, well-aligned anticapillary forceps to pick up the grids on the rimmed edge will minimize mechanical damage to the sections and support film.

50. The carbon coat will not only strengthen the coat, but will render this "reverse" side somewhat hydrophobic, facilitating the subsequent restriction of reagents to the section side of the grid.

51. It is sometimes helpful for later interpretation to collect sections consecutively.

52. Times can vary with block hardness and section thickness, and can be predetermined with test grids examined in the electron microscope for a loss of defined section edge.

53. Ideally, the carbon side of the grid remains dry during the entire immunostaining process. If the grid sinks at some point, it is best to proceed through each droplet immersed.

54. Antibody dilution is best determined by bracketing the dilution that gave the best light level localization.

References

1. Broker, T. R. and Chow, L. T. (1976) Electron Microscopy of Nucleic Acids, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.

2. Rosenberg, L., Hellman, W., and Kleinschmidt, A. K., (1970) Macromolecular models of proteinpolysaccharides from bovine nasal cartilage based on electron microscopic studies. J. Biol. Chem. 245, 4123-4130.

3. Heinegard, D., Lohmander, S., and Thyberg, J. (1978) Cartilage proteoglycan aggregates. Electron microscopic studies of native and fragmented molecules. Biochem. J. 175, 913-919.

4. Hascall, G. K. (1980) Cartilage proteoglycans: comparison of sectioned and spread whole molecules. J. Ultrastruct. Res. 70, 369.

5. Iozzo, R. V., Marroquin, R., and Wight, T. N. (1982) Analysis of proteoglycans by high-performance liquid chromatography. A rapid micromethod for the separation of proteoglycans from tissue and cell culture. Anal. Biochem. 126, 190-199.

6. Wight, T. N. and Hascall, V. C. (1983) Proteoglycans in primate arteries. III. Characterization of the proteoglycans synthesized by arterial smooth muscle cells in culture. J. Cell Biol. 96, 176-176.

7. Kapoor, R., Phelps, C. F., and Wight, T. N. (1986) Physical properties of chondroitin sulfate-dermatan sulfate proteoglycans from bovine aorta. Biochem. J. 240, 575-583.

8. Carlson, S. S. and Wight, T. N. (1987) Nerve terminal anchorage protein one (TAP-1) is a chondroitin sulfate proteoglycan: biochemical and electron microscopic characterization. J. Cell Biol. 105, 3075-3086.

9. Iwata, M., Wight, T. N., and Carlson, S. (1993) A brain extracellular matrix proteoglycan forms aggregates with hyaluronan. J. Biol. Chem. 268, 15,061-15,069.

10. Carlson, S. S., Iwata, M., and Wight, T. N. (1996) A chondroitin sulfate/keratan sulfate proteoglycan, PG-1000 forms complexes which are concentrated in the reticular laminae of electric organ basement membranes. Matrix Biol. 15, 281-292.

11. Movat, H. Z. (1955) Demonstration of all connective tissue elements in a single section. Arch. Pathol. 60, 289-295.

12. Schmidt, R. and Wirtla, J. (1996) Modification of movat pentachrome stain with improved reliability of elastin staining J. Histotechnol. 19, 325-327.

13. Musto, L. (1981) Improved iron-hematoxylin stain for elastic fibers. Stain Technol, 56, 185-187.

14. Sheenhan, D. C. and Hrapchak, B. B. (1980) Theory and practice of histotechnology, 2nd ed. C. V. Mosby, St Louis, MO.

15. Wight, T. N., Lara, S., Riessen, R., Le Baron, R., and Isner, J. (1997) Selective deposits of versican in the extracellular matrix of restenotic lesions from human peripheral arteries. Am. J. Pathol. 151, 963-973.

16. Tengblad, A. (1979) Affinity chromatography on immobilized hyaluronate and its application to the isolation of hyaluronate binding proteins from cartilage. Biochem. Biophys. Acta 5781, 281-289.

17. Ripellino, J. A., Klinger, M. M., Margolis, R. U., and Margolis, R. K. (1985) The hyaluronic acid binding region as a specific probe for the localization of hyaluronic acid in tissue sections. J. Histochem. Cytochem. 33, 1060-1066.

18. Knudson, C. B. and Toole, B. P. (1985) Fluorescent morphological probe for hyaluronate. J. Cell Biol. 100, 1753-1758.

19. Underhill, C., Nguyen, H., Shizari, M., and Culty, M., (1993) CD44 positive macrophages take up hyaluronan during lung development. Dev. Biol. 155, 324-336.

20. Riessen, R., Wight, T. N., Pastore, C., Henley, C., and Isner, J. M. (1996) Distributions of hyaluronan during extracellular remodeling in human restenotic arteries and balloon-injured rat carotid arteries. Circulation 93, 1141-1147.

21. Evanko, S., Raines, E. W., Ross, R., Gold, L., and Wight, T. N. (1998) Proteoglycan distribution in lesions of atherosclerosis depends on lesion severity, structural characteristics and the proximity of PDGF and TGF-p. Am. J. Pathol. 152, 533-546.

22. Evanko, S. P. and Wight, T. N. (1999) Intracellular localization of hyaluronan in proliferating cells. J. Histochem. Cytochem. 47, 1331-1341.

23. Evanko, S. P., Angello, J. C., and Wight, T. N. (1999) Formation of hyaluronan- and versican-rich pericellular matrix is required for proliferation and migration of vascular smooth muscle cells. Arterioscler. Thromb. Vasc. Biol. 19, 1004-1013.

24. Luft, J. H. (1971) Ruthenium red and violet. I. Chemistry, purification, methods of use, electron microscopy and mechanism of action. Anat. Rec. 171, 347-368.

25. Wight, T. N. and Ross, R. (1975) Proteoglycans in primate arteries. II. Synthesis and secretion of glycosaminoglycans by smooth muscle cells in vitro. J. Cell Biol. 67, 686.

26. Iozzo, R. V., Bolender, R. P., and Wight, T. N. (1982) Proteoglycan changes in the intercellular matrix of human colon carcinoma: an integrated biochemical and stereological analysis. Lab. Invest. 47, 124-138.

27. Snow, A. D., Bolender, R. P., Wight, T. N., and Clowes, A. W. (1989) Heparin modulates the composition of the extracellular matrix domain surrounding arterial smooth muscle cells. Am. J. Pathol. 137, 313-330.

28. Bolender, R. P., Hyde, D. M., and Dehoff, R. T. (1993) Lung morphometry: a new generation of tools and experiments for organ, tissue, cell and molecular biology. Am. J. Physiol. 265, 521-548.

29. Carlson, S. S. (1982) 103Ruthenium red, a reagent for detecting glycosaminoglycans at the nanogram level. Anal. Biochem. 122, 364-367.

30. Chen, K. and Wight,T. (1984) The ultrastructural identification of proteoglycans in arterial smooth muscle cell cultures using cationic dyes. J. Histochem. Cytochem. 32, 347-357.

31. Lark, M.W., Yeo, T-K., Mar, H., Lara, S., Hellstrom, I., Hellstrom, K. E., and Wight, T. N. (1988) Arterial chondroitin sulfate proteoglycan: localization with a monoclonal antibody. J. Histochem. Cytochem. 36, 1211-1221.

32. Gutierrez, P., O'Brien, K. D., Ferguson, M., Nikkari, S., Alpers, C. E., and Wight, T. N. (1997) Differences in the distribution of versican, decorin, and biglycan in atherosclerotic human coronary arteries. Cardiovasc. Pathol. 6, 271-278.

33. O'Brien, K. D., Alpers, C. E., Chiu, W., Hudkins, K., Wight, T. N. ,and Chait, A. (1998) A comparison of apolipoprotein and proteoglycan deposits in human coronary atherosclerotic plaques: colocalization of apolipoprotein E and biglycan. Circulation 98, 519-527.

34. Mar, H., Tsukada, T., Gown, A. M., Wight, T. N., and Baskin, D. G. (1987) Correlative light and electron microscopic immunocytochemistry on the same section with colloidal gold. J. Histochem. Cytochem. 35, 419-425.

35. LR White product instructions.

36. Erikson, P. Anderson, D., and Fisher, S. (1987) Use of UA en bloc to improve tissue preservation and labeling for immunoelectron microscopy. J. Elect. Micro. Tech. 5, 303-314.

37. Mar, H. and Wight, T.N. (1989) Correlative light and electron microscope immunocy-tochemistry on reembedded resin sections with colloidal gold, in: Colloidal Gold: Principles, Methods and Applications, Vol. 2, (Hyatt M. A., ed.), Academic, Orlando, FL.

38. Mar, H. and Wight, T.N. (1988) Colloidal gold immunostaining on deplasticized ultrathin section. J. Histochem. Cytochem. 36:1386-1395

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