Lc3b e5q2k mouse mab

Popliteal arteries were fixed with 4% paraformaldehyde for 24 h at room temperature, followed by procession by a serial alcohol gradient and embedment in paraffin wax blocks. The slides were incubated with primary antibodies that include GAB1 Rabbit mAb (#3232; Cell Signaling), LC3B (E5Q2K) Mouse mAb (#83506; Cell Signaling), and Human CD31/PECAM-1 Antibody Sheep mAb (#AF806-SP; R&D) overnight at 4°C, followed by the incubation of secondary antibodies that include donkey anti-rabbit IgG Alexa 488 (#A32790, Invitrogen, green), donkey anti-mouse IgG Alexa plus 647 (#A32787, Invitrogen, pink), and donkey anti-Sheep IgG Alexa 555 (#A-21436, Invitrogen, red). Nuclei were stained with DAPI (#D1306; Invitrogen, Paisley, United Kingdom). An epifluorescence microscope (#DM4000 M; Leica, Wetzlar, Germany) was used for image capture and analysis.

The cells were lysed in RIPA buffer (Solarbio, R0010). The protein concentration was determined using the BCA Protein Assay Kit (Meilunbio, MA0082). Equivalent amounts of protein were separated through SDS-polyacrylamide gels and electroblotted onto PVDF membranes (Bio-Rad, 1620177). After blocking using a 5% BSA Blocking Buffer (Solarbio, SW3015), the membranes were incubated with the primary antibody at 4 °C overnight. The antibodies used were AMPKα Antibody (Cell Signaling Technology, 2532, Danvers, MA, USA), Phospho-AMPKα Rabbit mAb (Cell Signaling Technology, 2535), mTOR Antibody (Cell Signaling Technology, 2972), Phospho-mTOR Antibody (Cell Signaling Technology, 2971), LC3B (E5Q2K) Mouse mAb (Cell Signaling Technology, 83506), SQSTM1/p62 Antibody (Cell Signaling Technology, 5114), and β-Actin Antibody (Cell Signaling Technology, 4967). Finally, the relevant protein was visualized through staining with an appropriate HRP-conjugated secondary antibody for 1 h and then enhanced with chemiluminescence. The expression of the protein was quantified using ImageJ software.