Routine Techniques of Histology
A. Light Microscopy (LM)
»» 1. Fixation
- fresh tissues are fixed in 10% formalin
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»» 2. Dehydration
- tissues are processed through a series of graded ethyl alcohol, such as 70, 80, 95, and 100%. |
»» 3. Clearing
with xylene, or benzene, or chloroform |
»» 4. Infiltration
-infiltrated with paraffin which is liquefied in oven at about 58C |
»» 5. Embedding
-embedded with hot liquid paraffin which is solidified at room temperature after embedding |
»» 6. Sectioning
-the paraffin blocks are sliced to sections of 5-10 microns in thickness with a sharp steel knife in the
microtome, and sections are picked up on slides |
»» 7. Staining
-dissolve the paraffin sections with xylene
-hydrate sections gradually from 100% alcohol to water
-stained with hematoxylin and eosin (H & E) |
»» 8. Observation
-with the light microscope |
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B. Transmission Electron Microscopy (TEM)
»» 1. Fixation
(prefixation and postfixation)
- tissue pieces are prefixed with 2-4% glutaraldehyde or a mixture of 2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.4, at 4C
-after washing in buffer, tissues are post fixed in 1% OsO4 in 0.1M cacodylate buffer, pH 7.4 at room temperature |
»» 2. Dehydration
-treated with increased concentration of ethyl alcohol (50%, 75%, 85%, 95%, and 100%) |
»» 3. intermediate solvent
»» 4. Infiltration
-with a mixture of propylene oxide and epoxy resin (epon 812) |
»» 5. Embedding
-with pure epon 812 which will be solidified in 60C oven for two days |
»» 6. Sectioning
-the epon block are cut to ultrathin sections of 60-90 nm in thickness with a glass or diamond knife in the ultramicrotome
-pick up ultrathin sections with grids |
»» 7. Double staining
-electron-opaque staining with uranyl acetate and lead citrate |
»» 8. Observation
-with the transmission electron microscope (TEM) |
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