Anti-Serotonin protocol (short version)

2) Rinse 3x with M9 (if needed), spinning down and removing supernatant

3) Fix overnight (ON) at 4^o C in 500 µl of 4% paraformaldehyde (PFA) / 1x PBS

4) Rinse 3x in PBSTx (0.5% Triton X-100 / 1x PBS).

5) Incubate ON at 37^o C in 500 µl of freshly made 5% beta-ME in TrisTx (1% TX-100 / 0.1 M Tris, pH 7.4)

6) Rinse 2x in TrisTx

7) Rinse 1x in collagenase buffer (1mM CaCl₂ / TrisTx)

8) Incubate 15 min - 8 hr at 37^o C in 2000 Units/ml Collagenase type IV (SIGMA C5138) in collagenase buffer - MONITOR digestion

9) Rinse 3x in PBSTx

10) Incubate for 1 hr (minimum) at RT in 100 µl of 1% BSA / PBSTx (or Ab Soln A)

11) Incubate ON at RT in 1:100 anti-serotonin in 1% BSA / PBSTx (or Ab Soln A)

12) Rinse 1x in PBSTx

13) Rinse 3-4x over 2-4 hr in 0.1% BSA/ PBSTx (or Ab Soln B)

14) Incubate ON at RT in the dark [OR 1-4 hr at 37^o C] in 50 µl of 1:100 TRITC-conjugated goat anti-rabbit antibody (or your preferred secondary antibody)

15) Rinse 1x in PBSTx

16) Rinse 3-4x over 1-2 hr in 0.1% BSA/ PBSTx (or Ab Soln B); Add DAPI to stain nuclei

17) Mount on agarose pad, coverslip and view with epiflourescence

Day 1 - Late afternoon/evening: Collect worms, fix ON at 4C.
Day 2 - Morning: rinse, put at 4C. Late afternoon/evening: b-ME treatment ON at 37C
Day 3 - Morning: rinse, do collagenase treatment, put in block at RT. Late afternoon/evening: Primary antibody ON at RT
Day 4 - Morning: rinse, add secondary antibody
Day 5 - Morning: rinse, mount and view
[OR Day 4 (in a hurry) - Morning: rinse, add secondary antibody, incubate 1-4 hr 37C, rinse, mount, view]

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