Anti-Serotonin protocol (short version)

Long version with detailed notes
Solutions for Anti-Serotonin staining
Last modified: 8 July 2014

1) Wash worms off plate with M9, put into 1.5 ml microcentrifuge tubes, spin down 30 sec in mini-centrifuge, remove supernatant

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

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

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

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

6) Rinse 2x in 1% TX-100/0.1 M Tris, pH 7.4

7) Rinse 1x in collagenase buffer (1mM CaCl2/1% TX-100/0.1 M Tris, pH 7.4)

8) Incubate 2-8 hr at 37o C in 2000 Units/ml Collagenase type IV (SIGMA C5138) in collagenase buffer

9) Rinse 3x in 0.5% Triton X-100/PBS

10) Incubate for 1 hr (minimum) at RT in 100 µl of 1% BSA/0.5% Triton X-100/PBS

11) Incubate ON at RT in 1:100 anti-serotonin in 1% BSA/0.5% Triton X-100/PBS

12) Rinse 2x in 0.5% Triton X-100/PBS

13) Incubate for 1 hr in 0.1% BSA/0.5% Triton X-100/PBS

14) Incubate 2-4 hr at 37o C with 50 µl of 1:100 TRITC-conjugated goat anti-rabbit antibody

15) Rinse 3x in 0.5% Triton X-100/PBS

16) Rinse for 1 hr in 0.1% BSA/0.5% Triton X-100/PBS (You can actually skip this step and look right away, with a slightly higher background; if so, do add some 0.1% BSA solution)

17) Mount on agar pad/coverslip and view with epiflourescence

Typical schedule for protocol:

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, Secondary antibody 1-2 hr at 37C, rinse, mount and view

If in a hurry, Days 3 & 4 can be combined - do primary antibody for 2 hr at 37C

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