Acetylated Tubulin cilia staining for X. tropicalis larvae
Modified from protocol by Basudha Basu
1. Gently place previously fixed and dehydrated embryos in baskets in 100% ethanol
See General Immunohistochemistry Protocol for information on embryo fixation and dehydration.
2. Rehydrate embryos in 25%, 50%, 75% and 100% PBS + 0.1% Tween (5 minutes
each)
- To make 100% PBS+0.1% PTween (PTW):
- add 100 ml 10X PBS + 900 ml DI water
- Tween is 0.1%, so add 1 ml (1 ml/1000ml PBS)
- in 50 ml Falcon tube:
i. 100% ethanol 75%(35ml) + water 25% (15ml), wash 5 min
ii. 100% ethanol 50%(25ml) + water 50% (25ml), wash 5 min
iii. 100% ethanol 25%(15ml) + PTW 75% (35ml), wash 5 min
iv. 100% PTW, wash 5 min
v. 100% PTW, wash 10 min
3. Block in 10% goat serum (because secondary is raised in goat) in PBT for 1 hour
- To dilute 100% goat serum to 10%: 200 microliters 100% goat serum +1800 microliters PBT = 2 ml , divide among 4 tubes, 500 ml each
- Put 1 ml 10% goat serum in 2059 Falcon tube, gently lower basket of embryos in
Add 1:2000 ac tub antibody in blocking buffer and incubate at 4 C overnight
ie. Add 1ul ac tub to 2000 ul PBT+ goat serum, vortex
5. Wash in PBT 3 times X 5 minutes
6. Add 1:500 Goat anti mouse Alexa Fluor 488 in blocking bu er and incubate at RT for 1 hour
ie. 2 ul Goat anti mouse: 1000 ul PBT, divide among 4 tubes, 500 ml each
7. Wash in PBT 3 times x 5 min</>
8. Mount for imaging
a. Place embryos on slide
b. Remove all liquid by wicking with Kim wipe
c. Add 80 - 100 ul mounting media(pro-long gold with DAPI)
d. Place cover slip carefully
Ordering info
Acetylated tubulin AB
Special order from Stockroom Sigma Prod. # T6793
Mounting Media- Invitrogen Prolong gold with DAPI
Product number : P36935
Alexa Fluor 488 goat anti mouse Ig, 0.5 ml
Special order from Stockroom
Invitrogen- Molecular Probes
Prod # A11001