Cy3 conjugated goat anti rabbit secondary antibody

Mφs on the climbing piece in 24-well culture plates were treated with or without bacterium infection or treated with relevant CM for 48 h. Then the cells were fixed with 4% paraformaldehyde for 20 min, washed with PBS, and permeabilized with 0.01% Triton X-100 for 10 min. After being washed with PBS, the cells were blocked with 10% goat serum for 30 min at room temperature and incubated with anti-CD86 monoclonal antibodies (Santa Cruz, USA) and anti-CD206 antibodies (Proteintech, China) at 4°C overnight. The next day, the cells were rinsed with PBS for clearing the primary antibody, and then incubated with Alexa Fluor 488-conjugated goat anti-mouse secondary antibody (Beyotime, China) or Cy3-conjugated goat anti-rabbit secondary antibody (Beyotime, China) at room temperature for 1 h in the dark, and then washed with PBS, and counterstained the nucleus with DAPI for 10 min. After washing again with PBS and mounting with antifade polyvinylpyrrolidone mounting medium (Beyotime, China), the fluorescent images were observed using confocal microscope (Lecia, Germany).

Chondrocytes were seeded in a 12-well plate and cultured until 80% confluence. After fixation and permeabilization, the cells were blocked with 5% Bovine Serum Albumin (BSA) for 1 h. Subsequently, the cells were respectively treated with primary antibodies against COL2 (1:500), GPX4 (1:500), STING (1:500), and TfR1 (1:200) at 4 °C overnight. Afterward, they were treated with Cy3-conjugated goat anti-rabbit secondary antibody (#A0516, Beyotime, Shanghai, China, 1:500) for 1 h at 37 °C in the dark. The cells were then subjected to a washing step and stained with DAPI (Boster, AR1177) for 5 min.
To investigate the colocalization of mitochondria with BNIP3 and Drp1, cells were incubated with a diluted Mito-Tracker Red CMXRos solution (#C1049B, Beyotime, Shanghai, China, 1:500) in the dark at 37 °C for 30 min. After fixation and permeabilization, the cells were then incubated with BNIP3 (1:200) and Drp1 (1:200) antibodies at 4 °C overnight. Subsequently, the cells were treated with FITC-conjugated goat anti-rabbit secondary antibody (A0562, Beyotime, Shanghai, China, 1:500) at 37 °C for 1.5 h in the dark. Following a wash with PBS and labeling with DAPI, fluorescence microscopy (Axio Observer 3; Carl Zeiss) was used to capture images and detect differences in the fluorescence expression of the corresponding proteins.

Ovaries tissues were fixed in 4% paraformaldehyde (Solarbio, Beijing, China) for 12 h and processed for standard 5 μm thick histological sections. Before staining, slides were incubated in 0.01 M sodium citrate at 96 °C for 10 min and blocked with BDT (3% BSA, 10% normal goat serum in TBS) for 45 min. and incubated with primary antibodies, rabbit anti-MVH antibody (1:150; Abcam, ab13840), rabbit anti-CX43 antibody (1:150) (Abcam, ab11370), rabbit anti-PPARα (Sangon, D161086, Shanghai, China), overnight at 4 °C. After careful washing with PBS, sections were incubated with CY3-conjugated goat anti-rabbit secondary antibody at 1:150 dilution (Beyotime, A0516, Nantong, China) at 37 °C for 30 min. Vectashield (Vector) was used to mount the slides. Images were taken under a fluorescence microscope (BX51; Olympus, Tokyo, Japan).

Intestinal tissues harvested from pigs were fixed in 4% PFA in PBS and incubated in 50% ethanol overnight. After fixation, tissues were embedded in paraffin, sectioned, and subjected to hematoxylin and eosin staining by standard procedures. For immunofluorescence analysis, samples were probed with rabbit anti-TGEV-N antibody (1:500; DA0224; Shanghai YouLong Biotech) for 30 min at 37°C. After three washes, samples were incubated with Cy3-conjugated goat anti-rabbit secondary antibody (Beyotime) and 4′,6-diamidino-2-phenylindole (DAPI) (Invitrogen). Images were obtained using a fluorescence microscope (Carl Zeiss).

After two steps of xylene dewaxing, slides were washed in a series of graded ethanol/water solutions. Then, slides were further incubated in 0.01 M sodium citrate at 96°C for 10 min. Samples were further blocked with BDT (3% BSA, 10% normal goat serum in TBS) for 45 min and incubated with rabbit anti-Caspase3 polyclonal antibody at a dilution of 1:150 (Abcam, ab2302, HongKong, China), rabbit anti-γH2AX polyclonal antibody at a dilution of 1:150 (Abcam, ab13840, HongKong, China), overnight at 4°C. After incubation, slides were washed three times with phosphate-buffered saline (PBS), then sections were incubated with CY3-conjugated goat anti-rabbit secondary antibody at a dilution of 1:150 (Beyotime, A0562, Nantong, China) at 37°C for 30 min. Vectashield (H-1000; Vector, Shanghai, China) was used to mount the slides. The proportion of Caspase3 and γH2AX positive cells was determined by calculating the number of positive cells in 5 different serial sections. All experiments were repeated at least three times independently.