Goat anti rabbit igg h l cross adsorbed secondary antibody alexa fluor 594

For cell samples, cells were fixed with 4% paraformaldehyde and permeabilized with 0.5% Triton X-100 PBS solution. After blocking with 5% bovine serum albumin (BSA, Beyotime, >99%), cells were incubated overnight at 4 °C with indicated primary antibodies (γ-H2AX, 1:200, Abcam; cTnT, 1:500, Abcam; β-actin, 1:1000, Proteintech; Alexa Fluor™ Plus 647 Phalloidin, 1:1000, ThermoFisher). Incubation with fluorescently coupled secondary antibodies (Goat anti-Rabbit IgG (H + L) Cross-Adsorbed Secondary Antibody Alexa Fluor™ 594, 1:1000, ThermoFisher; Goat anti-Mouse IgG (H + L) Cross-Adsorbed Secondary Antibody Alexa Fluor™ 488, 1:1000, ThermoFisher; Goat anti-Mouse IgG (H + L) Cross-Adsorbed Secondary Antibody Alexa Fluor™ 594, 1:1000, ThermoFisher) was followed by further washing in PBS. After washing, nuclei were visualized with 4,6-diamidino-2-phenylindole (DAPI, ThermoFisher). For histological samples, 7 µm tissue cryosections were washed with PBS to remove OCT. After fixation and permeabilization blocking, tissues were stained with the indicated primary antibody overnight. Afterward, the tissue was rinsed, stained with fluorescently coupled secondary antibodies for 1 h at room temperature, and then mounted with DAPI. The slides were visualized using a fluorescence microscope.

Cells were seeded at a density of 4.5 × 10³ cells/well in a 48-well plate. The next day, cells were serum-starved in DMEM supplemented with 0.5% FBS for 6 h before being cultured in full growth media for another 72 h. Cells were then washed with PBS and fixed with 4% paraformaldehyde for 20 min at room temperature before being washed again in PBS and blocked with staining buffer containing 1% BSA, 1% Tween 20, and 3% Triton-X in PBS. One hour later, cells were incubated with Ki67 antibodies (Abcam, UK) at 1:500 dilution overnight. Cells were washed with staining buffer before being incubated with Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 594 (Thermo Fischer Scientific, Waltham, MA, USA, cat#A11012) at 1:500 dilution and 4,6-diamidino-2-phenylindole (DAPI). Five random fields of 10× objective images were taken using a Nikon Ti-E fluorescence microscope (Nikon, Tokyo, Japan). Cell proliferation rate was calculated as the percentage of Ki67 positive cells of the total cell number per well as determined using the cell counter plugin of the Image J software (National Institutes of Health, Bethesda, MD, USA).

Tissue sections were fixed with 4% paraformaldehyde (PFA), permeabilized with 0.3% Triton X-100 for 30 min. Sections were washed three times with PBS, blocked with 10% normal goat serum and incubation with the following primary antibodies at 4°C overnight: anti-LC3II (Cell Signaling Technology, United States) and anti-LAMP1 (Santa Cruz Biotechnology, United States). The next day, they were incubated with Goat anti-Rabbit IgG (H + L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 594 (Thermo Fisher, United States) and IFKine Green Donkey Anti-Mouse IgG (Abbkine, China) for 1 h at 37°C, and treated with DAPI for 10 min. The sections were viewed by an inverted microscope.