Protocol by Ross Francis
Modified by Min-Ho Lee and Sudhir Nayak
1. Dissected gonads are usually fixed formaldehyde, methanol, acetone, or some combination (see Gonad Fixation). After fixation, wash gonads 2x in PBT before starting. Using a Pasteur pipette, transfer dissected, fixed gonads to a small [6 mm (ID) x 35mm] glass culture tube (Kimax brand). Spin 1 min at setting 3 and remove the supernatant.
2. Dilute primary and secondary antibodies in PBT with 30% goat serum (PBT+GS) and incubate as indicated below. Most anitobody incubations can be done in 100ul and as low as 50ul. Washes should be done with 500ul.
|
Treatment
|
Time
|
| PBT+GS with primary antibody | 8hrs to O/N |
| Wash 4x with PBT | 5min each |
| PBT+GS with secondary antibody | 4hrs to O/N |
| Wash 3x with PBT | 5min each |
| Wash 1x with 100 ng/ml DAPI in PBT | 5min |
| Place in anti-fade (DABCO) | 1min |
NOTES: The appropriate dilution of each primary and secondary antibody must be determined empirically. An initial blocking step is not required but may reduce background of some antibodies. If your primary antibody was generated in goat then you will need to use 1 mg/ml BSA in PBT instead of PBT+GS in all steps.
3. Using a drawn capillary pipette, transfer settled worms onto a large 2% agar pad that covers most of a slide. You will probably need to make several slides. After drawing off excess 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 – the image will improve as liquid evaporates and gonads become somewhat flattened (O/N). Slides can be stored at 4C for a week or more, particularly if sealed with nail polish or rubber cement around the periphery of the coverslip.
NOTE: If working with small numbers of animals, 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 can be very tedious.
Materials
PBS — Sambrook et al. Molecular Cloning book.
PBS/0.1% Tween 20 (PBT)
Blocking buffer; 30 % goat serum in PBT (PBT+GS) or 1 mg/ml BSA in PBT. Store at 4C once thawed. Good for weeks.
DAPI — 100 ug/ml stock solution. Dilute 1:1000 in PBS. Store at -20 good for years.
DABCO — 1% 1,4-diazobicyclo[2,2,2]-octane (DABCO) in 90% glycerol in PBS. Store at -20 good for years.