Antibody Staining of Dissected Gonads

互联网2013-09-05

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<center> Antibody Staining of Dissected Gonads - From R. Francis - -Adapted by Min-Ho Lee-</center>

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_~I 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~E 3 x 5 min or more and longer if you have background problem.
Secondary_Ab~I_Prior_to_incubation~E_preabsorbed_with_worm_formaldehyde~Hacetone_powder_at_least_4_hr_at_RT_or_overnight_at_4_oC_with_occasional_mixing_in_Blocking_buffer~E_spun_down_for_5_min_at_maximum_speed~E_take_super~E_and_incubate_at_least_4_hr_at_RT_or_overnight_at_4_oC_~AOvernight_at_4_oC_is_probably_best~B.~Kbr_~H~M~2~1~0 Wash_in_PBTw~E 3-4 x 5 min
~undefinedWash in PBS containing 100 ng/ml DAPI.
~undefinedWash in PBTw, once
~undefinedPlace 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