Anti sqstm1 p62 recombinant rabbit monoclonal antibody

Samples were extracted from the isolated P1-P14 cochlear SGNs. The sample proteins were resolved by SDS-PAGE on 12% polyacrylamide gels and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, USA). The membranes were blocked (Beyotime, China) under ambient temperature for 1 h and probed with the following primary antibodies at 4°C overnight: anti-MAP LC3II mouse monoclonal antibody (1 : 1000; Santa Cruz, sc-271625), anti-SQSTM1/P62 recombinant rabbit monoclonal antibody (1 : 1000; Abcam, ab109012, USA), anti-Beclin1 rabbit polyclonal antibody (1 : 1000; Abcam, ab62557, USA), anti-caspase3 rabbit polyclonal antibody (1 : 1000; Cell Signaling Technology, no. 9662, USA), anti-cleave-caspase3 rabbit monoclonal antibody (Cell Signaling Technology, no. 9664, 1 : 1000, USA), anti-Bcl2 rabbit monoclonal antibody (1 : 2000; Wanleibio, WL01556, China), and GAPDH (1 : 2000; Proteintech, 60004-1-lg, USA). After three washes, the membranes were incubated with a secondary antibody, HRP-labeled goat anti-rabbit IgG or anti-mouse IgG (1 : 1000; Beyotime, A0208/A0216, China), at 37°C for 1 h. Subsequently, the chemiluminescence reagent (Millipore, A : B = 1 : 1, USA) was utilized to develop the immunoreactive bands, and the images were analyzed using the Bio-Rad ChemiDoc XRS+ (Bio-Rad Company, USA). At least three independent experiments were carried out.

The paraffin-embedded tissue sections of P7 rat cochlea were blocked in a 1x PBS buffer containing 5% BSA (Sangon Biotech, C500626, China) and 0.3% Triton X-100 (Sigma, no. 30-5140, USA) at room temperature for 1 h, then incubated with the following primary antibodies at 4°C overnight: anti-MAP LC3II mouse monoclonal antibody (1 : 300; Santa Cruz, sc-271625, USA), anti-SQSTM1/P62 recombinant rabbit monoclonal antibody (1 : 200; Abcam, ab109012, USA), anti-Beclin1 rabbit polyclonal antibody (1 : 200; Abcam, ab62557, USA), anti-Bcl2 mouse monoclonal antibody (1 : 200; Servicebio, GB12318, China), anti-cleaved caspase3 rabbit polyclonal antibody (1 : 500; Servicebio, GB11009, China), and anti-caspase3 rabbit polyclonal antibody (1 : 500; Servicebio, GB11009-1, China). After three washes with 0.01 M PBS for 10 mins, the sample sections were incubated with the HRP-conjugated goat anti-rabbit/mouse IgG for IHC (ready to use) (1 : 300; Sangon Biotech, D110073, China) at room temperature for 40 min. After being incubated for 5 mins using a DAB Substrate kit (Sangon Biotech, E670033, China), the specimens were observed under a microscope (Olympus BX43, Tokyo, Japan) and images processed using Adobe Photoshop software.