This is a cached page for the URL (http://www.genetics.wustl.edu/tslab/gonad_antibody.html). To see the most recent version of this page, please click here.
Protocol Online is not affiliated with the authors of this page nor responsible for its content.
About Cache
Gonad Dissections
Antibody Staining of Dissected Gonads
- From R. Francis -
-Adapted by Min-Ho Lee-

Antibody staining  (in  Tubes)

1.  Using a pasteur pipette (drawn in flame and cut open), transfer worm carcasses with attached gonads to a small  (6 mm (O.D.) x 35mm) glass culture tube (Kimax brand).  Spin 1 min @ setting 2 in clinical benchtop centrifuge and remove super.  Wash once with PBS.

2.  Dilute primary and secondary antibodies in Blocking buffer and incubate as follows:
  *Blocking buffer 1 ­ 2 hr
  *Primary Ab :  4 hr or longer with occasional mixing (Overnight at room temp is probably best) in Blocking buffer.  Optimum dilution has to be decided empirically for each Ab.
  *Wash in PBTw,  3 x 5 min or more and longer if you have background problem.
  *Secondary Ab: Prior to incubation, preabsorbed with worm formaldehyde/acetone powder at least 4 hr at RT or overnight at 4 oC with occasional mixing in Blocking buffer, spun down for 5 min at maximum speed, take super, and incubate at least 4 hr at RT or overnight at 4 oC (Overnight at 4 oC is probably best).
  *Wash in PBTw,  3-4 x 5 min
*Wash in PBS containing 100 ng/ml DAPI.
*Wash in PBTw, once
*Place in 80% glycerol containing anti-fade reagent (e.g., Dapco) about 20 ­ 30 ul.
 Ab penetration is slower for dissected gonads than for embryos; hence, the longer incubation is better.

3.  Using a drawn and cut open capillary mouse pipette,  transfer settled worms onto a large 2% agar pad that covers most of a slide.  Will probably need to make several slides.  After drawing off exess liquid with a capillary, an eyelash hair (or finely drawn capillary) can be used to push gonads and intestines away from one another.  Cover with a large (24 x 50 mm) coverslip, taking care not to move the coverslip once in place.   Also do not seal the coverslip immediately -- image will improve as liquid evaporates and gonads become somewhat flattened.  Slides can be stored at 4 0 for a week or more, particularly if sealed with nail polish or rubber cement around the periphery of the coverslip.

For Smaller numbers of animals:
If working with small numbers of aniamls, all steps of the above steps can be done in a glass dish or depression slide.   As this requires that all steps be done while viewing in the dissecting scope it is tedious.

Materials

PBS -- Sambrook et al. Molecular Cloning book.
PBS/0.2 mM Levamisole (from 100 mM Levamisole stock).
PBS/0.1% Tween 20   (PBTw)
Blocking buffer; 30 % goat serum in PBTw  or  1 mg/ml BSA in PBTw.
3% Formaldehyde/0.1 M K2HPO4  (pH7.2). Prepared from sealed ampoules of 16% EM grade formaldehyde (EM Sciences). Freeze any excess.
formaldehyde/acetone powder ­ fix large quantity of mixed stage N2 with formaldehyde for 4 hrs and postfix with acetone for 10 min, wash with PBS several times, pass through French Press twice at 10, 000 psi, wash twice with PBS with 0.02 % sodium azide, and lyophilize the pellet to become powder.
fluorescent affinity-purified secondary Ab (from Chemicon or your preferred company).
DAPI -- 100 ug/ml stock solution. Dilute 1:1000 in PBS
Levamisole (Sigma) stock:  100 mM in dH20 (store @ -200C).
Methanol: 100% stock kept at -20 0C



Last revised 28 September 1997