Fitc conjugated anti rabbit igg

Differentiated myoblasts were cultivated in 10-mm dishes and used to perform immunostaining assay. The transfected cells were fixed in 4% formaldehyde for 20 min, followed the permeabilization with 0.5% Triton X-100 for 10 min and blocking with 1% bovine serum albumin (BSA) for 1 h. Besides, the next steps were incubation with primary antibody (anti-MyHC) (bs-9862R, Bioss, Beijing, China) (1:100) overnight at 4 °C, fluorescent secondary antibodies (FITC-conjugated anti-rabbit IgG) (1:200) at 37 °C for 1h and DAPI (Beyotime) for 5 min at room temperature. Ultimately, images were viewed using a confocal laser scanning microscopy (Zeiss LSM800, Göttingen, Germany).

The chemicals used in this study were crizotinib (Selleck Chemicals LLC, Houston, TX, USA), chloroquine (CQ), hydroxychloroquine (HCQ) (J&K chemical Ltd, Beijing, China) and 3-methyladenine (3-MA) (Sigma-Aldrich Corporation, St Louis, MO, USA). The primary antibodies used were against microtubule-associated protein 1 light chain 3 (LC-3), p62, Beclin-1, total or phospho-STAT3, AKT, MTOR, ERK, MET, EIF2AK2 and EIF2A and were from Cell Signaling Technology (Boston, MA, USA). The secondary antibodies were HRP-conjugated anti-rabbit IgG, anti-mouse IgG (Cell Signaling Technology) and FITC-conjugated anti-rabbit IgG (Beyotime, Nanjing, China).

Immunofluorescence staining was used to evaluate the expression of activated microglia. Rat brains were removed after cardiac perfusion with 200mL normal saline followed by 150mL 4% paraformaldehyde, fixed in 4% paraformaldehyde for 48h. Fixed brains were cut coronally through the needle entry site (identifiable on the brain surface), as well as 2mm anterior and 2mm posterior to that plane. The striatum was cut into 4μm thick each coronary slice. These slices were deparaffinized and incubated with 0.3% H₂O₂ in PBS. After blocking with 5% bovine serum albumin (BSA) serum, the sections were incubated with anti-Iba-1 antibody (diluted 1:100; Abcam) at 4°C overnight. On the second day, slices were covered with fluorescent-labeled secondary antibody FITC-conjugated anti-rabbit IgG (1:100, Beyotime), at 37°C for 30min, followed by DAPI (Beyotime) for 10min after washed in PBS. All sections were photographed and observed with a light microscope (Olympus/BX51, Tokyo, Japan).