Antibody Staining of Ovaries
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Antibody Staining of Ovaries
1. Dissect ovaries in EBR
2. Transfer ovaries into an Eppendorf tube and add 100 µl devitellinizing buffer and 600-µl heptane
3. Agitate gently for 10 minutes
4. Remove solution and rinse ovaries with PBS 3 times, then wash 3 times for 10 minutes each
5. It may help antibody penetration to dissect apart the egg chambers
6. Incubate ovaries in PBT for 10 minutes
7. Add monoclonal supernatant (either undiluted or 1:1 with PBT) or diluted polyclonal antiserum and incubate at 4 C overnight
8. Rinse with PBT 3 times, then wash 4 times for 15 minutes each
9. Add secondary antibody (usually 1:200 in PBT) and incubate 2 hours at room temperature
10. Rinse with PBT, then wash 4 times for 15 minutes each
11. Rinse twice with PBS
12. Add PBS:glycerol (1:1) and wait 20 minutes or so for ovaries to equilibrate
13. Dissect egg chambers in PBS:glycerol or antifade, coverslip, and look.
Devitellinizing Buffer
1 volume buffer B
1 volume 36% formaldehyde
4 volumes H2O
Buffer B
100mM KH2PO4/K2HPO4 (~25:20) pH 6.8
450mM KCl
150mM NaCl
20mM MgCl2. 6H2O
PBT
1X PBS
0.3% triton X100
0.5% BSA
10X PBS (g/l)
200 NaCl
5 KCl
5 KH2PO4
27.8 Na2HPO4.2H2O
EBR (Ringer's Solution)
Am. Nat. 70, 218-225.
Modified to use HEPES buffer instead of Tris.
10X EBR
mM 200 ml
NaCl 130 15 g
KCl 4.7 0.7 g
CaCl2 1.9 0.42 g (0.59 g CaCl2.2H2O)
HEPES, pH6.9 10 20 ml of 1 M
Filter sterilize and freeze in 25 ml aliquots.
Antifade
for 10 ml stock:
0.233 g DABCO (1,4-diazabicyclo(2.2.2.) octane) Sigma D2522
800 µl ddH2O
200 µl 1M Tris-HCL, pH 8.0
9 ml glycerol
Keep at 4 C covered in foil
Add 30 µl/slide and use large coverslip
Store slides at 4 C in the dark
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