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Plate cell at 104/chamber on 4-8 well chamber slide. No. of well plated depends on cells to be tested, no. Ab, no. of fixatives to try. Controls should be preimmune Ab and Ab with free peptide (for peptide antisera).

Possible fixatives:

a) 4% paraformalddehyde in PBS. Heat is 60C, add 1 drop/25ml 4M Na to dissolve. Cool to 4C. Check pH (~7). Fix cells for 10'

b) Methanol (RT) 2'

c) Acetone (RT) 2'

d) Acid alcohol (95% ethanol, 5% acetic acid) at - 20C for 5'

1. Aspirate media from cells and wash in PBS. Add fixatives as above, noting various times.

2. Aspirate and wash 1x PBS.

3. Post fixative permeabilzation 0.2% TX100/PBS 10' for PARAFORMALDEHYDE ONLY. Can also try methanol as a post-fix permeabalzing agent.

4. 1Ab 1:25 - 1:100 in 3% BSA/PBS

1 hr - O/N in humidified box at RT

6. Wash 3X PBS/3%BSA

7. 2 Biotin and Rabbit (or approp. species) 1:200 in 3% BSA/PBS for 45'+

8. Wash 3X PBS

9. 3C FITC Biotin-Avadin complex Vectastain . To prepare this complex:

Add this complex to the cells for 1 hr. Wash 3x PBS/3%BSA.

10. Drop of antifade , store at 4C in dark until viewing

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