2) Rinse 3x with M9, spinning down and removing supernatant
3) Fix overnight (ON) at 4 degrees C in 500 ul of 4% paraformaldehyde/PBS
4) Rinse 3x in 0.5% Triton X-100/PBS.
5) Incubate ON at 37 degrees C in 500 ul 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 37 degrees 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 at RT in 100 ul 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 37 degrees C with 50 ul of 1:100 rhodamine-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