Dr. John Kloetzel TEM Protocols
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Dr. John Kloetzel Protocols
Dr. John Kloetzel taught an electron microscopy class in1998 here at Keith R. Porter Imaging Facility, UMBC. The following is a transmission electron microscopy protocol written by Dr. Kloetzel for that class.Biol4221/622L
Fixation, Dehydration and Embedding of Tissues
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Background
The tissues to be fixed for this initial embedment will include mouse intestine, liver, and cardiac muscle. We will use these tissues because they have been widely studied and are well illustrated in the literature, so a ready standard of comparison exists. Also, together their cells contain most of the common cell organelles.[edit]
Some protozoan cells and plant tissues will also be fixed for comparison.
The tissue will be flooded with fixative solution (glutaraldehyde) in the animal immediately after sacrifice, removed and minced into small pieces, and placed in fresh fixative solution in glass vials for processing. Our tissue processing will be done at room temperature, except for an overnight buffer rinse n the cold, when higher temperatures would lead to increased tissue extraction. Following buffer rinses and “post-fixation” on buffered osmium tetroxide (OsO4), the tissue pieces are dehydrated (since the embedding medium [epoxy “plastic”] is not miscible with water). We will use Spurr’s (1969) low-viscosity epoxy resin for embedment. After passing through ethanol-epoxy mixtures, the tissue is soaked in the liquid epoxy mixture for an extended period (several hours to overnight) to permit through infiltration of the cells with resin. The tissue is then placed in a suitable embedding mold and monomeric epoxy is polymerized (“cured”,”hardened”) in an oven overnight. The cured “block” containing the embedded tissue will then be removed from the mold and be ready for trimming and thin sectioning on an ultramicrotome.
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Notes on the processing steps and solutions
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1. Fixative Solutions
The formula of the fixative we will use (made fresh as needed)Glutaraldehyde, purified 8% (EM grade) 3.75 ml
PIPES buffer, 0.1M, pH7.0 –7.4 4.0 ml
Sucrose stock (1M in DW) 1.0 ml
MgCl2 (0.1M and KCL (0.5M) stock in DW 1.0 ml
Distilled water (DW) 0.25 ml
Total 10.00 ml
( Final concentrations: 3% glutaraldehyde, 0.04M PIPES buffer, 0.1M sucrose, 50mM K+ and 10mM mg++).
A. Aldehyde fixatives
Primary fixation for EM now uses solutions of buffered glutaraldehyde (usually at 1-3%) almost universally. Sabatini et al.(1963) first promoted the use of a variety of aldehydes as fixatives; glutaraldehyde has prove most popular, sometimes in combination with formaldehyde (Karnovsky, 1965). As a dialdehyde (O=CH-CH2-CH2-CH2-CH=O), glutaraldehyde reacts at both ends with cell proteins (primary through interactions of the aldehydes with amino groups), effectively cross-linking proteins and maintaining cellular architecture (see Hopwood, 1972).
Formaldehyde (HCHO) is a ‘gentler’ fixative than glutaraldehyde; although it penetrates faster, it does not cross-link as effectively and fixation takes longer. Its primary uses are in the areas of enzyme and immuno cytochemistry, because it does not denature most enzymes and antigens as quickly as glutaraldehyde (see Berryman & Rodewald, 1990); Jamur et al., 1995; Sabatini et al, 1963). Ordinary commercial formaldehyde solutions (‘formalin’) contain methanol; such solutions are not used in EM. It is possible to make pure formaldehyde from powered para-formaldehyde (Karnovsky, 1965); purified formaldehyde for EM, sealed in ampules, is now available and easier to use.
Because of their reactive properties, it goes without saying that contact with these fixatives, and inhalation of their vapors, should be avoided. Use in fume hood.
B. Osmium tetroxide (OsO4)
The formula of the fixative we will use (stable):
OsO4 stock (4% in DW) 1 ml
PIPES Buffer, 0.1M, pH 6 to 7.4 2 ml
DW 1 ml
(Final concentrations: 1% OsO4, 50mM PIPES Buffer)
Osmium tetroxide was originally used as a ‘primary’ fixative (without prior aldehyde fixation); however, more material is leached out of the cells by the post-fixative rinses after OsO4 fixation than after aldehyde, so OsO4 is now used primarily as a second fixative (post-fixation) following glutaraldehyde or Karnovsk’s “pre-fixation”. The reason it is still used at all is that during fixation some osmium products bind to cell components, especially membranes, and ‘stain’ them for better microscopic appearance. OsO4 alxo reacts with some cell components (especially lipids) that aldehydes react with only poorly.
Traditionally, OsO4 fixation has been done in the pH 7.0 to 7.4 range. Maupin-Szamier & Pollard (1978) have shown that OsO4 at these pH’s can disrupt actin microfilament networks, and that OsO4 at pH 6.0 is less disruptive. Thus it could be wise to fix some tissue pieces with acidic OsO4 for comparison with traditional post-fixation. Time of exposure to OsO4, and it’s concentration, also are important in microfilament preservation; Maupin-Szamier & Pollard, 1978.
Under some conditions OsO4 and glutaraldehyde are mixed and used in a simultaneous double fixation (Hirsch & Fedorko, 1968). This combination fix seems most advantageous with single cell preparations example would be leukocytes or protozoans. These fixatives react with each other, so fix solution is made fresh and done at ice-bath temperatures.
OsO4 is ussally bought as a crystalline solid and made up to 4% in distilled water. It is fairly expensive, so it is used with as little waste as possible. It is also highly volatile and should be handled with caution, preferably in a fume hood. OsO4 vapors can fix your corneas and /or irritate your respiratory membranes if not treated with respect.
Store solutions in GLASS. OsO4 also reacts with corks, plastic lid liners, etc.
C. Buffers
Fixative solutions are usually buffered to near physiological pH (~pH7 to 7.4). We will use PIPES (Piperazine-N,N-bis[2-ethane sulfuric acid]), one of the “Good” buffers (Good et al.,1966) that is compatible with living systems (thus potentially less destructive?) and that seems to minimize protein and lipid extraction (Schiff & Gennaro, 1979). Other commonly used buffers for EM fixation include phosphate, cacodylate (similar in many ways to phosphate but based on arsenic and thus poisonous), veronal acetate and s-collidine.
D. Other additives
1.Sucrose- To maintain an osmolarity of the fixative solution near to physiological (~300 milliosmolar), inert agents are sometimes added. Most common is sucrose. The idea is to minimize disruption of cell shape and membrane integrity due to osmotic water fluxes (Bone & Denton, 1971). Sucrose will be used also in the post-fixative buffer rinses for the same reason. Once tissue has been exposed to OsO4, the osmotic integrity of cell membrane is lost and osmotic protectants are no longer needed. Even distilled water rinses can be used. 2. Salts- Empirically, inclusion of divalent cat ions in fixative solutions appears to help protect integrity during rinsing and dehydrating steps. The basis for this effect is not known, although some cross-linking or linkages involving the divalent ions are possible contributors. Calcium is more potent than magnesium in its membrane protective effects; however, as free Ca++ may destabilize microtubules, we will use magnesium. As the major free cat ion in living cytoplasm, it probably can’t hurt; there is a bit of “witchcraft” in all this anyway. KCL will be included in the fix, and both ions in the buffer rinses, following this same logic.
3.Miscellaneous additives- A number of other substances have been added to fix solutions in attempts to improve fixation quality generally, or preserve a certain type of structure specifically (see Boyles, 1982, for example of amine additions). One of the most interesting additives is tannic acid (TA), which in vitro has been shown to protect actin microfilaments from he degradative effects of OsO4 (Maupin et al, 1979). It also binds to and enhances the density and visualization of certain other cytoskeleton and membranous elements. Unfortunately, TA doesn’t penetrate living cell membranes well; therefore it is included in fix solutions in combination with low concentrations of detergents (digition, saponin) that aid its permeation into cells (see Maupin & Pollard, 1983). It has been reported that the use of TA ( together with uranyl acetate) following glurtaraldehyde-OsO4 fixation protects specimens from subsequent shrinkage during dehydration (Wollweber et al., 1981). TA-containing fixes are made fresh. T appears to be important to use certain low molecular weight TAs; most authors recommend the TA marketed by Mallinckrodt Chemicals, St. Louis: Type AR; code no. 1764).
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2. Post-fixation and en bloc staining with Uranyl acetate
Uranyl acetate has fixation properties for certain molecules (especially nucleic acids, not well fixed by aldehydes or OsO4[Terzakis, 1968; Hirsch & Fedorko, 1968] and phospholipids [Berryman and Rodewald, 1990; Erickson et al., 1987]. An important consequence of UAc’s reactivity with macromolecules is its ability to enhance contrast of cellular structures especially nucleic acids and membranes when exposed to pieces (blocks) of aldehyde-fixed tissue. Tissue pieces are soaked in aqueous UAc solutions as part of the dehydration sequence. This may help stablize the specimen against later shrinkage (Wollweber et al., 1981).[edit]
3 Dehydration
There are two basic methods used to eliminate water from the fixed tissue in the preparation for infiltration with epoxy plastic resins. One is the traditional “physical dehydration” in which tissue pieces are passed through a series of increasing concentrations of solvents most commonly ethanol or acetone until in pure or absolute solvent the water has all been replaced. In “chemical dehydration”, first used for EM by Muller and Jacks (1975), cell water is totally eliminated by soaking the wet fixed tissue in excess 2,2 dimethoxypropane (DMP). The hydrolysis of DMP by cell water, under slightly acidic conditions, proceeds:OCH3 O
| H+ ||
CH3-C-CH3 + H2O Æ 2CH3OH + CH3-C -CH3
|
OCH3
Thus using up cell water, leaving the tissue saturated with methanol, acetone and the remaining DMP in its place within a few minutes time. These solvents are miscible with the hydrophobic monomeric epoxy, so infiltration with the plastic mixture can begin, more quickly than with physical dehydration.
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4 Embedding resins
A.Epoxies:All epoxy embedding formulations are mixtures of :
1) monomeric resins containing epoxy bonds;
2) “hardeners” containing anhydride groups which react with epoxy bonds to form covalent bonds, cross-linking the reactants into polymeric form and thus hardening the previously liquid mixture; and
3) acceleralors, usually amines, to speed the polymerization reaction.
4) Flexibilizers, usually linear epoxides, added to soften and improve the sectioning qualities of the cured epoxy block.
A data sheet attached shows the chemical structure of many of the most common embedding ingredients.
A variety of epoxy resins are currently in use for EM embedding. Historically most common have been Araldite or Epon, or mixtures of the two (Mollenhauer,1964). Epon is no longer available but most EM suppliers market similar resins, “Poly/Bed 812 from Polysciences is one such epoxy from major EM material vendors.
We will use Spurr’s (1969) epoxy formulation because of its lower viscosity, which facilitates handling and speeds infiltration. (Vacuum treatment of the specimen during infiltration also aids epoxy penetration). It also is miscible with ethanol, which Epon and Araldrite are not, these need additional transitional solvents usually propylene oxide, between the ethanol’s and epoxy).
Epoxy ingredients are measured by weight into disposable beakers and mixed extremely thoroughly. Methods for avoiding atmospheric moisture contamination of the ingredients, which is especially important for the anhydride, and for storing excess resin in the freezer will be demonstrated.
Some people are sensitive to skin irritation by epoxies; one report (Ringo et al., 1982) suggests that they may even be mutagenic. We will minimize contact with th eepoxy ingredients whenever possible, using gloves and the hood. Resist the temptation of washing stray epoxy off your skin with ethanol or other solvents (as us old-timers used to do); these help the epoxies penetrate the skin. Wash thoroughly with soap and water after handling epoxy ingredients.
B. Acrylics
In recent years the use of acrylic resins such as LR White, LR Gold, and Lowicry, has increased dramatically. Because these resins are more hydrophilic than epoxies, they cause less denaturation of some macromolecules than epoxies, better preserving cellular penetration by aqueous reagents, e.g. antibody solutions, into the thin sections of embedded tissuses during protocols such as immunoelectron microscopy or in situ hybridization. See Berryman & Rodewald, 1990; Giammara,1993; Newman & Hobot, 1987.
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Tissue processing schedule
Tissues are exposed to fixative solution as quickly as possible, removed from their source, minced into 1-3mm pieces and immersed into fresh fixative. Perfusion fixation for animals forces fixative solution directly through the vascular system offering the quickest and the least disruptive fixation possible. In our schedule, the following steps will be carried out at room temperature unless cold ( 40C) is specified.
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Fixation
Fixation (glutaraldehyde) 0.5-2 Hr. @ R.T.:; can be longer in the coldBuffer rinses (40mM PIPES, 0.1M sucrose, 50mM KCL+10mM
MgCl2. 3 changes, 30 minutes to overnight; cold if need more time.
Post-fixation 1%OsO4, PIPES buffer 30 minutes – 2 Hr.
DW rinses. 2-3 changes, 2-5 minute each.
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Stain
En bloc staining 1% UAc in DW; protect from light. 30-60 minute.DW rinses. 2 changes, 2-5 minute each.
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Dehydration
Ethanol dehydration, 70%,95%, 3 changes of absolute. 5-10 minutes
each. Best to keep the times short.
OR
2,2-dimethoxypropane (acidified with 1 drop conc. Hcl/100DMP) 10min
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Infiltration
Spurr’s epoxy resin: Abs. EtOH 1:1(10-30 minutes, or until sample sinks)
Spurr’s :Abs. EtOH 5:1(10-30 minutes, or until sample sinks)
Spurr’s I (a few hrs. to overnight, R.T.)
Spurr’s II ( in labeled embedding mold capsules) (a few hrs. to overnight,
R.T.)
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Polymerization
Oven cure ~16hr, 65-700C.
In recent the use of microwave ovens has been exploited to greatly speed up the tissue fixation and embedding protocols typically used, in some cases permitting sectioning of fixed blocks of sample within 3-4 hours of initial fixation. Besides the obvious savings of time, there are reports that microwave processing may offer other advantages as well, such as improved preservation of antigens. See Giammara, 1993; Giberson et al., 1995, 1997; Jamur et al., 1995; Login & Dvorak,1994
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Epon-Spurr Recipe
Spurr FormulationErl 4206 10.0 gram
DER 736 6.0 gram
NSA 26.0 gram
GMAE 24 drops
Epon Formulation
Epon 812 25.0 gram (any Epon 812 substitute can be used)
DDSA 13.0 gram
NMA 12.0 gram
DMP 32 drops
Mix up the two resins separately then mix them together in a 1:1 ratio. Acetone, in our protocol, is used both as the dehydrating agent and the transition solvent. There is no need to use propylene oxide. This resin mixture can be cured in the microwave in an hour and 15 minutes or cured 24 hours in the oven at 700C.
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References
Berryman, M., and Rodewald (1990) An enhanced method for post-embedding immunocytochemical staining which preserves cell membranes. J. Histochem. Cytochem. 38:159-170.
Bone,Q., and E.J. Denton (1971) The osmotic effects of electron microscope fixatives. J. Cell Biol. 49: 571-581.
Boyles, J.K. (1982) A modified fixation for the preservation of microfilaments in the cells and isolated F-actin. J Cell. 95: 287a (Abstract).
Erickson, P.A., D.H. Anderson, and S.K. Fisher (1987) Use of uranyl acetate en bloc to improve tissue preservation and labeling for post-embedding immunoelectron microscopy. J. Electron Microsc. Tech. 5: 303.
Giammara, B.L. (1993) Microwave embedment for light and electron microscopy using epoxy resisns, LR White, and other polymers. Scanning 15:82-87.
Giberson, R., and R. Demaree, Jr., (1997) Microwave fixation: understanding the variables to achieve rapid reproducible results. Microsc. Res. Tech. 32:246-254.
Giberson, R., R. Demaree, Jr., & R. Nordhausen (1997) Four-hour processing of clinical/diagnostic specimens for electron microscopy using microwave technique. J. Vet. Diagn. Invest. 9:61-67.
Good, N.E. et al (1966) Hydrogen ion buffers for biological research. Biochem. 5:467-477.
Hirsch, J.G. and M. Fedorko (1968) Ultrastructure of human leukocytes after simultaneous fixation with glutaraldehyde and osmium tetroxide and “postfixation” in uranyl acetate. J. Cell Biol. 38:615-627.
Hopwood, D. (1972) Theoretical and practical aspects of glutaraldehyde fixation. Histochem. J. 4:267-304
Jamur, M.C. et al. (1995) Microwave fixation improves antigenicity of glutaraldehyde-sensitive antigens while preserving ultrastructure detail. J Histochem. Cytochem.43:307-311.
Karnovsky, M.J. (1965) A formaldehyde-glutaraldehyde fixative of high osmolality for use in electron microscopy. J. Cell Biol. 27:137a (Abstract).
Login, G.R., and A.M. Dvorak (1994) Methods of microwave fixation for microscopy. Progr. Histochem. Cytochem. 27:72-94.
Maupin-Szamier, P., and T.D. Pollard (1978) Actin filament destruction by osmium textroide. J. Cell Biol. 77:837-852
Maupin, P., and T.D. Pollard and J. Heuser (1979) Preservation of actin filaments with tannic acid containing fixatives in pure actin pellets, actin gels and intact cells. J. Cell Biol. 83:327a (Abstract).
Maupin, P., and T. Pollard (1983) Improved preservation and staining of HeLa cell actin filaments, clathrin-coated membranes, and other cytoplasmic structures by tannic acid-glutaraldehyde-saponin fixation. J. Cell Biol. 96:51-62
Mollenhauer, H.H. (1964) Plastic embedding mixtures for use in the electron microscopy. Stain Tech. 39:111-114
Muller, L.L., and T.J. jacks (1975). Rapid chemical dehydrtion of samples for electron microscopic examination. J. Histochem. Cytochem 23:107-110.
Newman, G.R. and J.Q. Hobot (1987) Modern acrylics for post-embedding immunostaining techniques. J. Histochem. Cytochem 35:971-981.
Ringo, D.L., E. Brennan and E. Cota-Robles (1982) Epoxy resins are mutagenic: implications for electron microscopists.
Sabatini, D.D., K. Bensch and R.J. Barrnett (1963) Cytochemistry and electron microscopy. The preservation of cellular ultrastructure and enzymatic activity by aldehyde fixation. J. Cell Biol. 17: 19-58
Schiff, R.I and J.F. Gennaro (1979) The role of the buffer in the fixation of biological specimens for transmission and scanning electron microscopy. Scanning 2:135-148.
Spurr, A.R. (1969) A low viscosity epoxy resin embedding medium for electron microscopy. J. Ultrastruct. Res. 26:31-43.
Terzakis, J.A. (1968) Uranyl acetate, a stain and a fixative. J. Ultrastruct. Res. 22:168-184.
Wollweber, L., R. Stracke & U. Gothe (1980) The use of a simple method to avoid cell shrinkage during SEM preparation. J. Microscopy 121:185-189.
