binds to centrosomes
From Pat
Monoclonal, Sigma clone GTU-88
Fix in paraformaldehyde or cold methanol
Dilute 1:100
From Drew Sigma T6557-100UL
- Working concentration is 1:5000
- Frozen stock is diluted 1:1 with glycerol
- 5 uL aliquots (in half-mL tubes) should be diluted:
- 1:50 in the tube: add 245 uL PBS-Tw-BSA to make 1:100 stock
- 1:50 for use (1 uL 1:100 antibody + 49 uL PBS-Tw-Az-BSA per coverslip)
Drew's Protocol:
- Rinse in PBS
- Fix in paraformaldehyde 15 minutes
- Rinse with PBS-Triton
- Rinse with PBS-Tween
- Rinse with PBS-BSA 1 hour
- Dilute Ab 1:5000 in BSA
- Incubate with Ab 2hr RT
- Rinse with PBS 3x
- Incubate with secondary Ab (in BSA)
- Rinse with PBS 3x
- Mount
Test Protocol:
- coverslips already fixed in paraglut, stored in PBS-Tw-Az 1
- 1 uL Ab (as supplied by Sigma) into 99 uL PBS-BSA (save!)
- 1 uL 1:100 Ab into 499 uL PBS-BSA (save for the rest of the slides if it works!)
- 50 uL onto coverslip - 2 hr RT incubation
- rinse with PBS-Tw-Az
- 50 uL Goat anti mouse 488
-
Results: some background fluorescence. Add a blocking step:
rinse 30 min in 2% BSA
rinse 2x in PBS-Tw-Az
Didn't make much difference. centrosomes are small and bright ↩︎
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