Nuclear marker dapi

Cellular proliferation was measured by nuclear incorporation of BrdU in the lungs of E18.5 embryos. BrdU (0.1 mg/g body mass) (B5002, Sigma Aldrich) was injected intraperitoneally into pregnant female mice at E18.5 and 2 h later the animals were anesthetized with isoflurane prior to euthanasia by cervical dislocation and removal of the embryos. Each embryo was weighted and processed for paraffin sections. Three-micrometer sections were deparaffinized and rehydrated, submitted to antigen retrieval treatment as described above, treated with 2N HCl for 45 min at 37 °C, neutralized with borate buffer (0.1 M pH 8.5 three times for 10 min each), washed with PBS and blocked with PBS, 0.2% Tween20, 5% BSA and 2% goat serum. After PBS washing, sections were immunostained overnight at 4 °C with a primary antibody for BrdU (1:2000, Accurate Chemical, OBT0030CX, diluted in PBS, 0.1% Tween20, 2% BSA and 2% goat serum). Sections were washed with PBS and incubated with the secondary antibody (1:500 diluted, goat anti-rat Alexa 488 from Jackson ImmunoResearch) and counterstained with nuclear marker DAPI (Sigma) for 1 h at RT. Preparations were then washed with PBS and mounted with ProLong Diamond anti-fading reagent (P36970, Life Technologies).

72 hpf embryos were fixed and used for immunohistochemistry procedure as outlined in Burke et al. [21 (link)]. In order to localize acetylated tubulin, serotonergic and synaptotagmin B positive cells we used 1:400 diluted mouse monoclonal anti-acetylated tubulin antibody (AcTub; T7451, Sigma-Aldrich), rabbit monoclonal anti-serotonin antibody (Ser; S55451E11, Sigma-Aldrich) and 1:50 diluted mouse monoclonal anti-synaptotagmin B antibody (1e11) [21 (link)], respectively. The fluorescent staining was developed using goat anti-Mouse IgG Alexa Fluor^® 488 conjugate (A-110010, Invitrogen) and Donkey anti-Rabbit Alexa Fluor^® 555 conjugate (A-31572, Invitrogen) (1:500) secondary antibodies. Lastly, the embryos were incubated with nuclear marker DAPI (Sigma-Aldrich) 1:10000 in PBT (phosphate-buffered saline; 0.1% Tween 20).