Antibody Staining of Ovaries

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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