5-HT (Serotonin) Goat Antibody

We use ImmunoStar 5-HT goat antibody (ID #20079) to stain brain tissue of a marine fish. We combine it with fluorescent-labeled secondary antibodies for visualization under epifluorescence microscopy. This antibody produces a robust signal with minimal background and clear staining of somata and processes. Our neuroanatomy results with ImmunoStar goat anti-serotonin are identical to those we found using another well known antibody from another manufacturer made in rabbit. Brain tissue is from animals transcardially perfused with Ringer's solution followed by 4% paraformaldehyde in phosphate buffer (0.1M, pH 7.2). Tissue is cryosectioned at 25 microns.Our procedure is as follows: 1. Rehydrate tissue in PBS (0.1M phosphate buffer saline, pH 7.2) for 10 minutes twice. 2. Immerse tissue in PBST+DS-5% (PBS with 5% donkey serum and 0.3% Triton-X) for 1 hour. 3. Apply goat 5-HT antiserum diluted 1:500 in PBST+DS-5% and incubate at room temperature overnight. 4. Wash tissue with PBS+DS-0.5% (0.5% donkey serum solution, no Triton-X) for 10 minutes five times. 5. Apply secondary antiserum diluted 1:400 in PBST+DS-5% and incubate at room temperature for 2 hours. 6. Wash with PBS for 10 minutes six times. 7. Mount slides and view.

I find ImmunoStar's goat anti-serotonin (#20079) to be just as reliable as the rabbit anti-serotonin antibody. I've used this on sectioned brain tissue from moths, flies, cockroaches and preying mantids. I always get very even penetration of the antibody although I have never tried thicker than 150um sections of brain embedded in agarose blocks. Below is my protocol: Day 1: Overnight (O/N) at 4oC in 4% paraformaldehyde in 0.01 Phosphate Buffered Saline (PBS). Day 2: Wash tissue 3x 5 min in PBS. Embed in 5% agarose and section at 50-150um. Wash 4x 15 min in PBS with .5% Triton X-100 (PBST). Block 1 hr in PBST with 2% Bovine Serum Albumin (BSA). O/N at room temperature (RT) in 1:1000 goat anti-5HT in PBST with 5mM sodium azide (PBSAT) with 2% BSA and 1% Triton X-100. Day 3: Wash 4x 30 min PBST. Block 1hr 2% BSA in PBST. O/N at RT in 1:1000 donkey anti-goat Alexa 546 (2% BSA in PBSAT). Day 4: Wash 2x 15min PBST. Wash 2x 15min PBS. Wash 1x 15min in 60% glycerol. Mount in 80% glycerol.

I utilized the antibody in formalin-fixed, free-floating mouse brain tissue at a concentration of 1:1000. For secondary detection I utilized Invitrogen Alexa-Fluor-conjugated secondary antibodies at 1:200. The 5HT antibody always yielded great staining and we were very pleased with it. Indeed, we utilized this antibody for studies of dorsal raphe 5HT neurons as reported in our recent publication in Cell Metabolism (Lam, Leinnintger et al 2011, Cell Metab 13(5): 584-91).