Anti synapsin

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).

Worms were fixed in Carnoy's and processed as in Beane et al. (2012). Primary antibodies: anti‐arrestin, 1:10,000 (kind gift from M. Levin, Tufts University, Medford, MA); anti‐synapsin, 1:50 (Developmental Studies Hybridoma Bank 3C11, Iowa City, IA), and anti‐phospho‐histone‐H3 (Sigma/Millipore 04‐817, St. Louis, MO), 1:25. Secondary antibodies: for arrestin and synapsin, goat anti‐mouse Alexa 488, 1:400 (Molecular Probes A‐11001, Eugene, OR); for H3P, goat anti‐rabbit horseradish peroxidase (Invitrogen 65‐6120, Carlsbad, CA) with TSA Cy3‐Tyramide amplification (Perkin Elmer, 1:50, Waltham, MA).

Immunostaining was carried out as previously reported.
³² (link),
³³ (link) The planarians 1–5 mm in length used for experience were killed with 5% NAC in PBS (phosphate buffered saline) for 5 min at room temperature, washed three times with PBST (phosphate buffered saline containing 0.1% TritonX‐100) at RT and fixed in PBST containing 4% paraformaldehyde for 2–4 h at 4°C. Then, worms were placed in 100% methanol for 1 h at −20°C and treated with 1% SDS in PBST for 10 min at RT. After rinsed in PBST, the samples were blocked with 10% goat serum in PBST for 2 h at 4°C or RT. They were then incubated with primary anti‐synapsin (1:100; Developmental Studies Hybridoma Bank) or anti‐H3P antibodies (1:250; Millipore, 05‐817R) overnight at 4°C. The specimens were washed four times with PBST for 30 min per wash before adding the secondary antibody goat anti‐mouse Alexa Fluor488 (1:500) and goat anti‐rabbit Alexa Fluor568 (1:500). Stained planarians were visualized using a fluorescence microscope (Olympus, Tokyo, Japan).