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Cryosection |
Negative stain |
TEM cytochemistry |
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TEM freeze substitution |
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isolate sample |
isolate sample |
isolate sample |
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isolate sample |
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mount on coated grids |
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mount on coated grids |
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fix with buffered aldehydes and/or cryoprotect |
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gentle fix with buffered aldehydes |
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rapid freeze |
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rapid freeze |
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rinse |
rinse |
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cytochemical reactions |
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transfer to -80C solutions of acetone with or without aldehyde and osmium for 3 days |
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rinse |
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Postfix with buffered aldehyde, followed by buffered osmium |
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rinse |
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slowly warm to room temp |
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dehydrate to 100% alcohol |
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rinse with 100% acetone |
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embed in resin |
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embed in resin or LRW/Lowicryl |
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polymerize |
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polymerize |
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thick and thin slice at -100C |
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thick and thin slice |
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thick and thin slice |
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thaw slices and incubate in antibody solutions |
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if necessary, incubate in antibody solutions |
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stain |
stain with UA |
stain UA & Pb |
stain UA & Pb |
stain in UA |
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stain UA (& Pb) |
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mount on sample stubs |
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evaporator coat with platinum and carbon inside machine |
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evaporator coat with platinum and carbon |
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sputter coat (or view at low kV) |
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remove sample, thaw to room temp., and digest sample if necessary |
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digest sample if necessary |
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