Alexa fluor 594 labeled goat anti rabbit igg h l

After 2, 4, and 8 weeks, left sciatic nerves were harvested from rats in each group for histochemical staining. The nerves were fixed in 4% paraformaldehyde for 24 h, cryoprotected in 30% sucrose overnight at 4 °C, and then sectioned on a cryostat. The sections were attached to poly-lysine-precoated glass slides (Superfrost*/Plus microscope slides, Fisher Scientific) and prepared for staining. The tissues were washed with PBS three times for 15 min each, incubated with 0.1% Triton X-100 for 1 hour at room temperature, and blocking solution was applied (0.1% BSA), followed by incubation with the primary antibodies anti-NF200 protein mouse monoclonal antibody (Santa Cruz, 1:200) and anti-S100 protein rabbit monoclonal antibody (Santa Cruz, 1:200) at 4 °C overnight. The secondary antibodiesAlexa Fluor 488-labeled goat anti-mouse IgG (H + L) (1:500, Invitrogen) and Alexa Fluor 594-labeled goat anti-rabbit IgG (H + L) (1:500, Invitrogen) were applied to tissue samples for 2 hours at room temperature in the dark. This step was followed by washing in PBS. All of the slides were also stained with DAPI (1:500) at room temperature for 15 min away from light and were examined with a fluorescent microscope (BX-60; Olympus).

The antibodies and drugs used in this study included rabbit monoclonal anti-Oct-1 antibody (Abcam, ab178869), rabbit monoclonal anti-HCF-1 antibody (Abcam, ab289975), mouse monoclonal anti-VP16 antibody (Santa Cruz, sc7545), mouse monoclonal anti-β-actin antibody (Sino Biological, 1000166), rabbit monoclonal anti-GAPDH antibody (Abways Technology, AB0037), mouse monoclonal anti-Histone-H3 antibody (Sino Biological, 100005-MM01), rabbit polyclonal antibody anti-TSG101 (Proteintech, 28283-I-AP), mouse monoclonal anti-HSV-1-gD antibody (Santa Cruz, sc21719), mouse monoclonal anti-ICP0 antibody (Santa Cruz, 13,118), Alexa Fluor 594-labeled goat anti-rabbit IgG (H + L; Invitrogen, 2165334), Alexa Fluor plus 488-labeled goat anti-mouse IgG (H + L; Invitrogen, A32723), goat anti-mouse IgG-HRP (Invitrogen, 31430), goat anti-rabbit IgG (H + L)-HRP (Invitrogen, 32460), and protease inhibitor cocktail (Thermo Scientific, EO0492).

5637 cells or T24 cells were incubated with anti-TRPV2 antibody (1:100 dilution, ACC-139, Alomone Labs) at 4 °C for 30 min. Subsequently, cells were washed and incubated with Alexa Fluor™ 594-labeled goat anti-rabbit IgG (H + L) (1:100 dilution, A-11037, Invitrogen, CA, USA) for 1 h. After staining, cells were washed and suspended in PBS for flow cytometric analysis. For detection of intracellular Ca²⁺ concentration in 5637 cells or T24 cells by flow cytometry, Fluo-3 AM (S1056, Beyotime, Jiangsu, China) was used according to the manufacturer's instructions.