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INFORMATION FOR

Acetylated Tubulin cilia staining for X. tropicalis larvae

Modified from protocol by Basudha Basu

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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