Anti cht1 monoclonal antibody mouse anti rat igg

Animals were anesthetized with 4% isoflurane and sacrificed. Bilateral colonic DRGs (T12 to S1) were dissected, fixed in 4% paraformaldehyde, and embedded in paraffin. All the specimens were cut into 5 μm thick sections. After deparaffinization and rehydration, antigen retrieval was performed at microwave oven for 15 minutes in citrate buffer (0.01 mM, pH = 6.0). After blocking with 2% BSA and washing, sections were then incubated in the anti-CHT1 monoclonal antibody (mouse anti-rat IgG, 1 : 200, Santa Cruz Biotechnology, CA, USA) overnight at 4°C. PBS instead of the primary antibody served as negative control. After washing three times, sections were then incubated with HRP-conjugated antibody (goat anti-mouse IgG, 1 : 200, Boster, Wuhan, China) for 30 minutes and stained by 3,3′-diaminobenzidine (DAB) chromogenic reagent. Finally, counterstaining was performed with hematoxylin. The signals obtained from the labeling cells were detected via Olympus BX53 microscopy (CCD DP80).

Equal protein (25 μg) from bilateral colonic DRGs (T12 to S1) was separated by electrophoresis on 10% SDS-PAGE and then transferred to nitrocellulose membranes. After blocking with 2% BSA and washing, membranes were incubated with anti-CHT1 monoclonal antibody (mouse anti-rat IgG, 1 : 200, Santa Cruz, CA, USA) at 4°C overnight. Membranes were subsequently incubated for 2 hours with HRP-conjugated IgG antibody (goat anti-mouse IgG, 1 : 5000, Servicebio, Wuhan, China). Data was presented as band density which was normalized relative to GAPDH (1 : 1000, Servicebio, Wuhan, China).