Routine Techniques of Histology 




 
A. Light Microscopy (LM)

»»  1. Fixation

- fresh tissues are fixed in 10% formalin

»»  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

-with prophylene oxide

»»  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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