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how to do a histology study, Dye, mixtures, TEM, acetic acid, microscopy,…

- - - - clearing
        
        embedding
        
        sectioning
        
        paraffin removal
        
        staining
        
        mounting and covering
        
        xylene or toluene resolve it then are replaced with water
        
        light microscopy (microtome)
        
        electron microscopy (diamond or plateglass knife)
        
        embeding in celloitin is longer but less shrinking (for eye and brain)
        
        replace aldohol by xylene or toluene
        
        why? parafin and alcohol arent miscible
      - embedding in gelatin doesnt need dehydration doesnt need alcohol
      - why? paraffin hydrophobe
    - - crosslinking fixatives
        
        aldehydes
        
        formaldehyde
        
        binds to the active part of the protein
        
        cause little distortion, penetrate rappidly
        
        hardens slowly but doesnt protect from shrinking
        
        glutaraldehyde
        
        forme dimeres et trimeres
        
        react with amino acid grp
        
        large mol so peut pas entrer ds des tissus epais mtl formaldehyde
        
        not recommended for paraffin
        
        mostly used is 0.5 a 3% de glutaraldehyde et formaldehyde
        
        oxidizing agents (chromic acid, osmium tetroxide, potassium dichromate, potassium permanganate)
        
        osmium tetroxide
        
        dont penetrate deep
        
        only used as 2nd fixative in electron microscopy
        
        used as stain also since 1985
        
        light microscopy used for unsaturated lipid, myelin and makes them insoluble in water and fat solvant
      - denaturing fixatives
        
        alcohol
      - others
        
        picric acid
        
        mercuric chloride
    - - by freezing (fluid N2 or dry ice)
      - in microwave
  - - - heat kill and adhere the organism to the slide