Goat anti 5ht

C. elegans serotonin antibody staining was performed using the tube fixation protocol (McIntire et al., 1992 (link)). Briefly, synchronized young adult hermaphrodites were fixed in 4% paraformaldehyde (PFA) for 18 hr, with β-mercapto-ethanol for another 18 hr, with 1 mg/ml collagenase (Sigma Aldrich, Merk, Darmstadt, Germany) for 90 min and incubated for 24 hr with rabbit anti-5HT antibody (1:5000; Sigma Aldrich). Alexa 555-conjugated donkey anti-rabbit (1:500; Molecular probes) was used as secondary antibody.
For mouse immunohistochemistry, freshly isolated E11.5 embryos from C57Bl/6JRccHsd were fixed by immersion in 4% PFA. Rabbit anti-Sall2 (1:100; Sigma Aldrich), goat anti-Brn2 (1:100; Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-5HT (1:5000; Sigma Aldrich) and goat anti-5HT (1:200; Abcam, Cambridge, UK) antibodies were used. As secondary antibodies Alexa 555-conjugated donkey anti-rabbit and anti-goat, and Alexa 488-conjugated donkey anti-rabbit and anti-goat were used (1:600; Molecular probes, Invitrogen, Eugene, OR). Immunofluorescence samples were analyzed and photographed using a confocal TCS-SP8 Leica microscope.

To detect HCN2 immunofluorescence in murine intestine, mice were euthanized with an overdose of pentobarbital and were prefixed through transcardiac perfusion with saline followed by 4% paraformaldehyde (PFA). Intestinal tissues were removed and post-fixed with 4% PFA overnight at 4°C. The fixed tissues were transferred to 30% sucrose for dehydration until the tissue had sank to the bottom of centrifuge tube. Then the specimens were embedded with OCT. For immunofluorescence staining, 10 μm-thick frozen sections were cut. Tissue sections were washed four times with PBS and blocked with 3% BSA and 10% normal donkey serum in PBS contained 1% TritonX-100 for 1 h at room temperature. Then tissue sections were incubated with rabbit anti-HCN2 (1:1000, Alomone, Israel) or goat anti-5-HT (1:2000, Abcam, Cambridge, MA, United States) at 4°C for 48 h. After four rinses with PBS, the sections were incubated with donkey anti-rabbit Alexa Fluor 488 secondary antibody (1:1000, Invitrogen, Eugene, OR, United States) or donkey anti-goat Alexa Fluor 568 secondary antibody (1:1000, Invitrogen, Eugene, OR, United States) for 2 h at room temperature. After washing with PBS, the sections were mounted onto glass slides with mounting medium, then observed and photographed using fluorescent microscope (Leica DM 2500, Germany).