Anti h3p

Whole-mount immunostaining was performed as previously described [58 (link)]. In each experiment, ten animals were used in each group. Animals were killed in PBS with 5% NAC for 5 min and washed three times with PBST at room temperature. Then, the animals were fixed in 4% paraformaldehyde for 2–4 h at 4 °C and incubated in 100% methanol for 1 h at −20 °C. Thereafter, the animals were blocked with 10% goat serum in PBST for 2–4 h at 4 °C and incubated with primary anti-synapsin (1:100; Developmental Studies Hybridoma Bank, Shanghai, China) or anti-H3p (1:250; Millipore, 05-817R, MA, USA) antibodies overnight at 4 °C. After six times of washing with PBST, the animals were labeled with goat anti-mouse Alexa Fluor 488 (1:500; Invitrogen, 673781, Shanghai, China) or goat anti-rabbit Alexa Fluor 568 (1:500; Invitrogen, 11036). Finally, the animals were observed by NIS element software (version 4.2.0, Olympus, IX73P1F, Tokyo, Japan).

Colorimetric and FISHs were performed as previously described (Pearson et al., 2009 (link); King and Newmark, 2013 (link)). Following fluorescent or NBT/BCIP development, animals were incubated with anti-acetylated-Tubulin antibody (1:1000, Cell Signaling, Danvers, MA), anti-H3P (1:1000, Millipore), or a rabbit antiserum recognized unknown epitope to visualize the lumen of PT (1:500). Primary antibodies were detected with either Alexa-conjugated anti-rabbit antibodies (1:1000; Abcam) or HRP-conjugated anti-rabbit antibodies (1:1000; Jackson ImmunoResearch). NBT/BCIP developed whole-mount in situ specimens were mounted in mounting media containing 75% glycerol and 2 M urea. Fluorescent whole-mount in situ specimens were mounted in modified ScaleA2 containing 20% glycerol, 2.5% DABCO and 4 M urea (Hama et al., 2011 (link)). For cryosectioning, fluorescently stained whole-mounted animals were fixed overnight in 4% paraformaldehyde (in PBS) at 4°C, washed three times in PBS, equilibrated in 30% sucrose, frozen in OCT, and cryosectioned (10–20 μm).

Whole-mount immunostaining was performed as previously described [52 (link)]. In brief, the animals were killed with 5% NAC in phosphate-buffered saline (PBS) for 6 min at room temperature and washed four times with PBS containing 0.1% TritonX-100 (PBST). Then, the animals were fixed in PBST containing 4% paraformaldehyde. Next, the animals were blocked with 10% goat serum in PBST for 2 to 4 h at 4 °C and incubated with primary anti-H3P (1:500; Millipore, Burlington, MA, USA, 05-817R) or anti-synapsin (1:100–1:500 Developmental Studies Hybridoma Bank) antibodies overnight. The secondary antibodies include goat anti-rabbit Alexa Fluor 568 (1:500; Invitrogen, Waltham, MA, USA, 11036) for anti-H3P, and goat anti-mouse Alexa Fluor 488 (1:500; Invitrogen, 673781) for anti-synapsin. Digital pictures were collected using NIS element software (version 4.2.0, Nikon, Tokyo, Japan).