5-HT (Serotonin) Rabbit Antibody

I have used this antibody to stain for 5HT positive cells in rat spinal cord after rehabilitation or transplantation therapies. I have always gotten consistent and beautiful staining at dilutions 1:80000 for DAB and 1:20000 for IF. Tissue preparation for rat tissue: TTranscardial perfusion using 4% paraformaldehyde, post fixed overnight in 4% para, and cryoprotected in 30% sucrose for 3 days. Freeze tissue, sectioned (at 40 micron), and use immediately within days. Indirect Immunofluorescence staining protocol: DAY 1 1. Wash 1x PBST for 15 min 2. Blocking solution (1x PBS + 0.3% Triton X100, 3% goat serum) for 1 hr at RT 3. Incubate tissue sections to primary antiserum (rabbit anti-5HT, ImmunoStar #20080) diluted in blocking buffer overnight @ 4C. DAY 2 5. Wash tissue sections 3 x 10 min with 1xPBS. 6. Incubate tissue sections with secondary antibody (e.g. goat anti-rabbit-Alexa Fl 488, Jackson ImmunoResearch Laboratories, Inc., diluted 1:500 in PBS) for 1-2 hrs in the dark at room temperature. 7. Wash tissue sections 3 x 15 min with 1x PBS in the dark. 8. Coverslip with vectashield and store at RT.

I have used the 5-HT Rabbit antibody (# 20080) for both my honors and masters project in multiple species including sea urchin (Lytechinus variegatus), polychaete (Capitella telata), and two cephalopods (Sepia officinalis and Loligo pealeii). I use my antibodies in immunohistochemistry in whole mount preparations in 1:200 dilutions. This antibody reliably stains the cell bodies and axons of the central nervous system in all my invertebrate preps. I love the consistency of staining I get across my study specimen, and the reliability of the product.

We have used this antibody to stain for 5HT positive cells in tissue sections (rat brain stem and spinal cords tissue samples) or in cultured mouse ventral mesencephalic cells. We have always gotten consistent and beautiful staining at dilutions 1:1000 - 1:5000. Tissue preparation for rat tissue: TTranscardial perfusion with 4% paraformaldehyde in 0.1M sodium phosphate buffer (PBS), pH 7.4, post fixed overnight in 4% para overnight, and cryoprotected in 30% sucrose until they sink. Freeze tissue, sectioned (at 16 micron), and thaw mounted on to gelatin coated slides. Samples are stored in -80C until use. Indirect Immunofluorescence staining protocol: DAY 1 1. Remove slides from -80C freezer and equilibrate to room temperature for at least 2 hrs. 2. Immerse slides in 1x PBST for 15 min 3. Remove slides from PBST and expose tissue sections to blocking solution (1x PBS + 0.3% Triton X100, 3% goat serum) for 1 hr at RT. 4.Incubate tissue sections to primary antiserum (rabbit anti-5HT, ImmunoStar #20080) diluted in blocking buffer overnight @ 4C. DAY 2 5. Wick off antiserum and wash tissue sections 3 x 10 min with 1xPBS. 6. Incubate tissue sections with secondary antibody (e.g. goat anti-rabbit-Alexa Fl 488, Jackson ImmunoResearch Laboratories, Inc., diluted 1:500 in PBS) for 1-2 hrs in the dark at room temperature. After incubation with secondary antibody, wash tissue sections 3 x 15 min with 1x PBS in the dark. 7. Counterstain with DAPI. 8. Coverslip with glycerol gel and store at RT. For in vitro cultured cells: Fix samples with 2% paraformaldehyde for 20 min at room temperature. Follow the exact protocol for staining.