Fitc conjugated f4 80

Flow cytometry analysis was performed to quantify immune cells (CD45 positive), macrophage (F4/80 positive), and NLRP3. Mouse hearts were subjected to myocardial infarction and treated with or without RSV for 5 days. Hearts were harvested, washed with cold PBS, and then quickly minced into small pieces (1-3 mm³) on ice. Heart tissues were digested by 0.1% collagenase II and 2.4 U/mL dispase II in PBS for 30 min at 37°C, and culture medium containing 15% serum was added to terminate cell digestion. Culture medium was filtered and centrifuged with 3000 rpm, and cells were resuspended in PBS. The CD16/32 antibody was used for blocking for 30 min, and cells were incubated with Per-cy5.5-conjugated CD45 (BD Biosciences) and FITC-conjugated F4/80 (BD Biosciences) for 30 min according to the manufacturer's instruction. To stain intracellular NLRP3, cells were fixed and permeabilized by Fixation/Permeabilization Solution (BD Cytofix/Cytoperm™ Plus). After adding the NLRP3 antibody (ABCAM), cells were incubated with Alexa Fluor 633-Goat-Anti-Rabbit-IgG (H-L) secondary antibody for 15 min. A flow cytometer (Beckman, Miami) was used for fluorescence detection, and flowjo_v10 was used for data analysis.

For immunohistochemistry (IHC), cryosections in the thickness of 10 µm were prepared from OCT-embedded tissue blocks and stained with indicated antibodies. Primary antibodies utilized for IHC examination included α-smooth muscle actin (α-SMA) (Sigma, USA), FITC-conjugated F4/80 (BD, Biosciences, USA). The sections were examined and the photomicrographs were recorded using fluorescent microscope (DM6000B, Leica, Germany).