4.13.1. Tissue fixation
- Dissect out individual tissues.
- Wash tissues with cold PBS 2-3 times.
- Fix tissues
in freshly made 4% paraformaldehyde in 100mM PBS (pH 7.0) overnight at 4oC.
- Rinse in
nuclease-free water three times.
- Store tissues in
70% ethanol at 4oC until further use.
- For tissue
dehydration, carry out successive incubation in ethanol (70%, 95% and 100%) and
xylol (2 x 5 min each).
- Embed in paraffin.
- Cut Paraffin
sections into 2-5 micron thick.
- Mount on
poly-L-lysinated slides which are to be
stored at 4°C overnight.
- 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.