Antibody staining for Drosophila ovaries
Horacio Frydman- (Reference: Frydman and Spradling, Development 2001) Wieschaus' Lab 5-31




 All dissections and solutions should be at Room Temperature

 1) Dissect ovaries at in 1x Grace's (not supplemented). Leave your tissue on Grace's for no more than 20 minutes. The sooner you fix, the better will be the cytoskeleton morphology

 2) Fix 20 minutes  in Grace's Fix

 3) Do 3 quick washes in PBT to remove fix, then wash 30 min. in PBT.  Gently rock ovaries (eg. on a "nutator") during all wash steps; they do not need to rock at other times.

 4.  Incubate 30 min. in PBT-NGS.

 5.  Incubate 2-4 hrs at RT or overnight at 4ºC in primary antibody, diluted into PBT-NGS.

 6.  Remove antibody and save.  (Primary antibodies can usually be re-used 2-3x or more, depending on the antibody)  Wash 2 hrs. in several changes of PBT-BSA.

 7.  Incubate 30 min. in PBT-BSA-NGS (PBANG)

 8.  Incubate 2-4 hrs at RT or overnight at 4ºC in fluorescently-labeled secondary antibody, diluted into PBANG

 9.  Wash 3x with PBT.

 10.  Wash 2 hrs. (or overnight at 4_C) in PBT-BSA

 11.  Wash briefly 1-2x in PBS and mount in PROLONG GOLD or your favorite antifade

 Good Luck

Solutions:

 Grace's Fix:
-1 volume 16% Formaldehyde (EM GRADE, with no Methanol); 3 volumes Grace's Insect Cell Culture Medium (not supplemented, Gibco-BRL  Cat No. 11590-056); 0.2 % Triton X-100

 Solution                        2ml     5ml     10ml
 16% PF (EM grade)   500µl   1.25ml  2.5ml
 10% Triton X-100           40µl    100µl   200µl
 Graces                     1500 µl 3.75ml  7.5ml

PBT
-1X PBS
 -O.2% Triton X-100

PBT-BSA
-PBT plus 0.2% BSA

PBANG
-Above plus 5% Normal goat serum