Antibody Staining

Adapted from Ross Francis

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Reagents:

• 0.1% PBT
• 30% Goat Serum (Once Thawed Good for few weeks.)
• Primary antibody Diluted in 30% Goat Serum
• Secondary antibody Diluted in 30% Goat Serum
• Diluted Dapi Solution
• 1% DABCO (Stored in -20° C)

Materials:

• Drawn Pippette
• 1.5 mL microcentrifuge tubes (VWR, 14231-062)
• Eppendorf MiniSpin Plus (Microcentrifuge)
• Foil
• Agar Pads

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Important Notes:

• Failure to wash away methanol will result in a high background when analyzing data.
• Avoid centrifuging these samples. Doing so may destroy the germ line. If necessary, centrifuge at 1000 rpm.
• The appropriate dilution of the primary and secondary antibody must be determined empirically. To reduce the background of the antibody, it is suggested, but not required to block the samples. To block samples, simply add 100 μL of 30% Goat Serum and place in room temperature for 30 minutes.
• If the primary antibody was generated in goat, then use 1mg/ml BSA instead of PBT instead of goat serum.
• Keep samples covered in foil after addition of secondary antibody.

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Procedure:

1. Dissect worms as described in the Dissection Protocol.
2. Fix the worms in 3% paraformaldehyde as described in the Gonad Fixation Protocol.
3. Add the primary antibody for 8 hours to overnight.
4. Gently add 500 μL 0.1% PBT to the side of the microcentrifuge to wash the primary antibody. Allow the samples to sit for 5 minutes. Note: Avoid centrifuging these samples. Doing so may destroy the germ line. If necessary, centrifuge at 1000 rpm.
5. Repeat wash 2 more times. Again, allow the samples to sit for 5 minutes each time.
6. Add the secondary antibody for 4 hours to overnight. Note: Optimum time is 4 hours. Longer exposure will increase the background of samples.
7. Gently add 500 μL 0.1% PBT to the side of the microcentrifuge to wash away the secondary antibody. Note: Avoid centrifuging these samples. Doing so may destroy the germ line. If necessary, centrifuge at 1000 rpm.
8. Cover samples with foil to prevent breakdown of fluorochrome.
9. Add 500 μL of Diluted Dapi Solution. Let sit for 5 minutes in the dark.
10. Wash sample with 500 μL 0.1% PBT. Note: Avoid centrifuging these samples. Doing so may destroy the germ line. If necessary, centrifuge at 1000 rpm.
11. Add 30 μL of 1% DABCO to the solution.
12. Using a wide bore pipette, gently pipette the solution and worms up and down several times. Then, deposit the worms onto agar pads.
13. Use a drawn pipette to remove the excess solution from the pads before applying a cover slip to each pad. Note: Gently place the cover slip corner by corner to avoid air bubbles.