Anti h3p

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).

Whole-mount immunostaining was performed as previously described [52 (link)]. The animals were killed with 5% NAC in phosphate-buffered saline (PBS) for 5 min at room temperature and washed four times with PBS containing 0.1% TritonX-100 (PBST). The animals were then fixed in PBST containing 4% paraformaldehyde, and cold 100% MeOH for 1 h. Next, animals were blocked with 10% goat serum in PBST for 2 to 4 h at 4 °C and incubated with primary anti-synapsin (1:100–1:500 Developmental Studies Hybridoma Bank) or anti-H3P (1:500; Millipore, Burlington, MA, USA, 05-817R) 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 (Nikon, Tokyo, Japan).