To be able to view a biological sample in the electron microscope it must first be stabilized or "fixed", preferrably in a way that the ultrastructure of the cells or tissue remain as close to the living material as possible. The choice of fixative depends on the purpose of your study. While finding the right fixation conditions is often a matter of trial and error, standard protocols can be sufficient for many purposes. Below are some of the most common chemicals used in routine TEM fixation.
Glutaraldehyde reacts with many nucleophiles in the cell (most commonly amines). It produces irreversible cross-linking networks throughout the cytoplasm in seconds to minutes. The reaction results in a drop in pH from a significant release of protons, making adequate buffering important. Note that a too high concentration of glutaraldehyde can inhibit the formation of rapid cross-links.
Formaldehyde cross-links amino groups of proteins. The reaction is much slower than that of glutaraldehyde, normally using a 2-8% solution you need to fix for at least 2 hours at RT. Due to the drop in pH during the reaction of formaldehyde and amino groups adequate buffering is important.
Glutaraldehyde-formaldehyde mixtures. The original paper by Karnovsky (1965) recommends a mixture of 4% glutaraldehyde and 6% formaldehyde. Other empirically determined mixtures are widely used for both structural and immunocytochemical studies. We routinely use a mixture of 2% formaldehyde and 2.5 % glutaraldehyde in 0.1 M Sodium Cacodylate buffer, pH 7.4.
Acrolein is a very toxic and highly volatile reactive aldehyde that's mostly been used in mixtures with glutaraldehyde and/or formaldehyde. Cross-links proteins as rapidly as glutaraldehyde.
Osmium tetroxide is normally used as a secondary fixative after formaldehyde/glutaraldehyde. It reacts with unsaturated acyl chains of membrane lipids and nucleophiles like amino and sulphhydryl groups. While its main purpose is contrasting it also increases the retention of lipids in the tissue.
Tannic Acid is a mixture of polyglycol anions that preserve tissue via non-covalent interactions. Can be used after primary fixation with an aldehydes to enhance contrast and preserve antigenicity. Makes a good substitute for Osmium tetroxide when used in combination with 1% Uranyl Acetate.
Embedding in resins
The routine method for examining biological samples in TEM is to embed the material in plastic and cut ultrathin (~60-80nm) sections. The choice of resin depends on the type of sample and the purpose of your study. Listed below are some of the more commonly used resins.
Epon or Epon-Araldite mixtures are the most widely used resins for electron microscopy. Epon is excellent for morphological studies but not a good choice for most immunocytochemistry purposes. The sample has to be completely dehydrated in protein denaturing solvents (like propylene oxide or acetone) before infiltration and polymerization has to take place at temperatures around 50-60° C.
Spurr’s Resin is a Low Viscosity mixture which provides rapid infiltration of tissues. It's easy to prepare and mixes rapidly. This resin is compatible with ethanol so no change to propylene oxide is needed prior to infiltration, although we recommend it. Polymerization at 60°C is recommended.
LR White and LR Gold are blends of hydrophilic and acrylic monomers that rapidly penetrate tissue because of their low viscosity. LR Gold is cured by UV-light in the cold while LR White can be cold cured (using an accelerator) or heat cured. The hydrophilic nature of these resins makes them usable for immunocytochemistry. Lowicryl K4M, HM20, K11M and HM23 are highly cross-linked acrylate and methacrylate based embedding media designed for use over a wide range of embedding conditions. They are usable for embedding at low temperatures and for immunocytochemistry.
Routine Protocol for Embedding in Resin
Our standard fixative is a mixture of 1.25% formaldehyde, 2.5 % glutaraldehyde and 0.03% picric acid in 0.1 M Sodium cacodylate buffer, pH 7.4.
OR a mixture of 2.5% glutaraldehyde and 2% formaldehyde in 0.1M Sodium cacodylate buffer, pH 7.4.
Fixation of cells: Add fixative (2x concentration) 1:1 to the cell media. Leave for 1 hour at RT. If cells grow in suspension, spin them down in the fixative at 3000rpm for 3 minutes. Leave for 1 hour at RT in the fixative.
Fixation of tissue: Perfusion fixation is preferred. For this we recommend using 2% formaldehyde and 2.5 % glutaraldehyde in 0.1 M Sodium Cacodylate buffer, pH 7.4. After perfusion, dissect out the tissue of interest, immerse in fresh fixative and cut into 1-2 mm cubes - leave to fix at least 2 hours at RT.
Bring your fixed samples to the EM lab in the fixative at this point. We will follow the protocol below:
Wash in cacodylate buffer 3x
1% Osmiumtetroxide/1.5% Potassiumferrocyanide (in H2O) leave for 1 hour at RT
Wash in H2O (or in malelate buffer pH 5.15) 3x
1% Uranyl Acetate in H2O (or in maleate buffer) for 30 minutes - 1 hr
Wash in H2O 3x
Dehydration: 70% EtOH 15 min/ 90% EtOH 15 min/ 100% EtOH 2x15 min
Propyleneoxide 1 hour
Infiltration: Epon mixed 1:1 with propyleneoxide ON at 4 C
Move samples to embedding mold filled with freshly mixed Epon
Allow sample to sink, move to oven for polymerization
Leave to polymerize for 24-48 hours at 60°C
Osmium Free Method for Resin embedding (Tannic Acid/Uranyl Acetate method)
1. Fix in PFA/ Picric acid as above. For immuno EM chose a fixative that works well for your antigen. (4% formaldehyde+0.01-0.1% glutaraldehyde is a good start)
Do the following processing on ice until 100% Ethanol:
2. Freshly made 1% Tannic Acid in Maleate Buffer (MB) for 40 min, then rinse 2x in MB
3. In the dark: 1% Uranyl Acetate in MB for 40 min, then rinse twice in MB
4. Dehydrate 5 min each: 50%/70% Ethanol
5. 1% PPD(p-phenylenediamine) in 70% Ethanol for 15 min
6. Rinse twice in 70% Ethanol
7. Dehydrate 5 min each: 80%/95%/2x100% Ethanol
8. Resin infiltration (LR white or other hydrophilic resin)
9. Polymerization at 55-60° C for 2-3 hrs.
Preparation of ultrathin frozen sections (Tokayasu)
Fixation of cells in culture: Attached cells: first remove cells from the dish with 5mM EDTA/PBS using a transfer pipet with a wide opening. Pipet up and down to release the cells from the dish as quickly as possible. Add cell suspension to a microfuge tube containing an equal volume 2x fixative (8% formaldehyde) in 0.2 M sodium phosphate buffer pH 7.4. (not PBS) and spin down to a pellet (~3000rpm for 3 min). Take off supernatant and add layer fresh 4%formaldehyde in 0.1M Sodium Phosphate buffer on top of the pellet - do not resuspend the cells. Leave pellet to fix for 2 hrs at RT! It's important not to resuspend the cells after they have been pelleted, this way the pellet will crosslink and stay together better in the sucrose infiltration step.
Cryoprotection: Small pieces (~2mm) of tissue or cell pellet is placed in a 200ul drop of 2.3 M Sucrose in PBS containing 0.15M glycine. Move to a fresh drop after a few minutes, do 3 drops for a total of 15 min at RT (or overnight at 4 °C).
Freezing: Sucrose infiltrated tissue/cells are mounted on an aluminum pin, excess sucrose is removed with a filterpaper and the pin is plunged into liquid nitrogen. Specimens can be stored for years under liquid nitrogen in cryo vials.
Sectioning: Ultrathin sections are cut at -120°C with a cryo-diamond knife, or at -90°C with a glass knife. Sections are picked up from the knife with a loop dipped a 2:1 mixture of 2.3M sucrose and 2% Methylcellulose and transferred to a formvar/carbon coated copper grid. Grids are left floating section side down on PBS or placed on 2% gelatin in a small petri dish and stored in the fridge until the immunogold labeling can be done.
Immunogold labeling procedure for Ultrathin Frozen Sections
If you stored the sections on 2% gelatin, warm up gelatin to 37degrees for 20 minutes, the gelatin will melt and you can transfer the grids to drops of PBS. Wash on 4 drops of PBS before blocking in 1% BSA.
1. Blocking, 1% BSA in PBS (15 min)
2. 1° Antibody diluted in 1%BSA (30 min RT)
3. Wash, 4 drops PBS (15 min)
4. If 1° antibodies with weak binding capacity for protein A (mouse, rat, goat, sheep) are used you need to use a bridging antibody (e.g. rabbit anti mouse) (30 min) followed by washing in 4 drops of PBS before the Protein A-gold step.
5. Protein A-gold (PAG) in 1% BSA (20 min)
6. Wash, 4 drops PBS (15 min)
In case of double labeling, fix in 1% Glutaraldehyde for 5 min, followed by quenching in 4 drops of 0.15 M Glycine/PBS and repeat points 2-7 with another size PAG.
7. Wash, 4 drops PBS (15 min)
8. Wash, 6 drops water (20 min)
Contrasting procedure: Mix 9 parts 2% methyl cellulose with 1 part 3% aqueous uranyl acetate. Float grids on the mixture on ice for 10 min. Pick up with a 3.5mm loop and remove excess liquid with a filterpaper (Whatman #1). Allow to dry before gently removing the grid from the loop and examining in the TEM.
The carbon film becomes hydrophobic with time resulting in uneven spreading of the sample and the stain. The best result will be obtained if the grid surface is made hydrophilic prior to use. This can be done in our table top glow discharge unit (negative charge for 20-30 seconds at 25mA).
The sample is diluted in water (if possible) and adsorbed onto a glow-discharged carbon or formvar/carbon coated grid. 1 minute is usually enough, depending on the concentration of your sample. Once the specimen has been adsorbed on to the film surface, the excess liquid is blotted off using a filterpaper (Whatman #1) and the grid is floated on a small drop (~5 µl) of staining solution (most commonly 0.75% uranyl formate, 1% uranyl acetate or 1-2% PTA). After 20 seconds the excess stain is blotted off and the sample is air dried briefly before it’s examined in the TEM. If your sample is in a buffer that contains phosphate or salts, wash the grid on a droplet of water before staining with uranyl acetate. (Uranyl will make a salt with the phosphate)
Routine Protocol for SEM
1. Fixation 1-2 hrs in 2% glutaraldehyde in 0.1M Sodium cacodylate buffer, pH 7.4
2. Rinse 3 X 10-15 min 30-45 min in 0.1M sodium cacodylate buffer, pH 7.4
3. Post-Fix 1-2 hrs in 1% Osmium tetroxide in water
4. Rinse 3 X 5 min in water
50% ETOH 5 min
70% ETOH 5 min
95% ETOH 10 min
100% ETOH 10 min
100% ETOH 10 min
6. Critical Point Dry 45-60 min or dry using HMDS.
7. Mount specimens onto stubs by using conductive carbon tape, silver paint or paste. If you use paint or paste, it must be dried overnight before going into the Sputter Coater.
8. Sputter Coating. The thickness you use can vary between 1- 10 nanometers.
9. SEM Imaging