Rabbit anti peripherin

Plated cells were fixed in 4% paraformaldehyde (PFA; Fisher Scientific) for 20 minutes at room temperature and rinsed with PBS. Nonspecific labeling was blocked and the cells permeabilized with 5% normal goat serum (Millipore) and/or 5% normal donkey serum (Millipore) and 0.2% Triton X-100 (Sigma) in PBS for 30 minutes at room temperature. Cells were rinsed with PBS and then incubated with primary antibodies for one hour at room temperature or overnight at 4°C. Cells were subsequently labeled with the appropriate fluorescently-tagged secondary antibodies. Hoechst nuclear dye was used to label nuclei. Primary antibodies used were rabbit anti-Peripherin (Millipore, AB1530), mouse anti-βIII Tubulin (Tuj1, Promega, G7121), rabbit anti-NTRK1 (Millipore, 06-574), rabbit anti-TRPV1 (Novus Biologicals, NBP1-97417), mouse anti-GFAP (Cell Signaling, 3670), rabbit anti-Parvalbumin (Calbiochem, PC255L), mouse anti SMI32R (Covance, SMI-32R), guinea pig anti-VGlut1 (Millipore, AB5905), and myelin protein zero (ProteinTech, 10572-1-AP). Secondary antibodies included donkey anti-mouse AF488 (Invitrogen, A21202), goat anti-rabbit RhoRed (Invitrogen, R6394), and goat anti-guinea pig AF488 (Life Technologies, A11073).

The cultures were rinsed with phosphate-buffered saline (PBS) and then were fixed with 3.7% formaldehyde (Wako) in PBS for 10 min at room temperature (RT). After fixation, the cells were permeabilized with PBS containing 0.2% Tween-20 (Wako Pure Chemical Industries) for 10 min, and then were blocked with PBS containing 4% Block Ace (DS Pharma Biomedical) and 0.2% Tween-20 for 1 h at RT. Primary antibodies diluted in the Can Get Signal Solution 1 (Toyobo) were incubated with the cells overnight at 4°C. Next day, the cells were washed three times with PBS containing 0.2% Tween-20, and then were incubated for 2 h at RT with the secondary antibodies (anti-mouse Alexa Fluor-488 and anti-rabbit Alexa Fluor-555; 1:1000; Life Technologies) diluted in the Can Get Signal 2 solution (Toyobo). The primary antibodies used in this study were as follows: mouse anti-class III beta-tubulin (TUJ1; 1:1000; Covance), mouse anti-P75NTR (neurotrophin receptor) (1:200; Advanced Targeting Systems), mouse anti-HNK-1 (1:500; Sigma-Aldrich), mouse anti-cTnT (cardiac Trophonin T) (1:500; Abcam), rabbit anti-Synapsin-1 (1:1000; Millipore), and rabbit anti-Peripherin (1:1000; Millipore). To stain the nuclei, 0.2 μg/ml Hoechst 33342 (Dojindo Molecular Technologies) was added to the Can Get Signal 2 solution.

Cells cultured on coverslips were fixed in 4% PFA (Sigma) in PBS (pH7.4) for 15min on ice, washed twice with PBS and incubated at room temperature for one hour in blocking solution (0.1% gelatin, 1% FBS, 0.5% Triton X-100 (BDH) in PBS). Fixed cells were incubated overnight at 4°C with primary antibodies diluted in blocking buffer. Cell monolayers were washed (4X10min) in PBS-Tr (0.1% Triton X-100 in PBS) and incubated for two hours with appropriate secondary antibody and DAPI diluted in blocking buffer. Unbound secondary antibody was removed by PBS-Tr washes (4X10 min), followed by two PBS washes before mounting in Mowiol (Sigma). Stained cells were and imaged on a Leica DMRB microscope. Primary antibodies used for immunocytochemistry and their dilutions are as follows: mouse anti-nestin (Rat401, DSHB) 1:50, mouse anti-NF-M (2H3, DSHB) 1:5, mouse anti-Islet (40.2D6, DSHB) 1:5, rabbit anti-peripherin (Millipore) 1:1000, rabbit anti-MAP2 (Millipore), goat anti-CHAT (Millipore) 1:200, rabbit anti-GAD1 (Millipore) 1:500, rabbit anti-Tyrosine Hydroxylase (Epitomics) 1:500, goat anti-mouse Alexa Fluor 488 (Life Technologies) 1:2000, goat anti-mouse Alexa Fluor 568 (Life Technologies) 1:2000, goat anti-rabbit Alexa Fluor 488 (Life Technologies) 1:2000, goat anti-rabbit Alexa Fluor 594 (Life Technologies) 1:2000. DAPI (Sigma) 1:2000.