4.13.1. Tissue fixation

  1. Dissect out individual tissues.
  2. Wash tissues with cold PBS 2-3 times. 
  3. Fix tissues in freshly made 4% paraformaldehyde in 100mM PBS (pH 7.0) overnight at 4oC.
  4. Rinse in nuclease-free water three times.
  5. Store tissues in 70% ethanol at 4oC until further use.
  6. For tissue dehydration, carry out successive incubation in ethanol (70%, 95% and 100%) and xylol (2 x 5 min each).
  7. Embed in paraffin.
  8. Cut Paraffin sections into 2-5 micron thick.
  9. Mount on poly-L-lysinated slides which are to be stored at 4°C overnight.
  10. To rehydrate the sections prior to hybridization:
    1. Carry out descending concentration of ethanol (100%, 95% and 70%).
    2. Dewax in xylol.
    3. Treat with proteinase K (10 ug/ml) for 30 min.
    4. Acetylate with 0.33% (v/v) acetic anhydride in 0.1 M triethanolamine-HCl (pH 8.0) for 10 min.
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