Rat anti nlrp3

Primary antibodies used in this study were rabbit anti-Iba-1 (1:300; Wako Chemicals, Tokyo, Japan), rat anti-nlrp3 (1:300; R&D Systems, Minneapolis, MN, USA), and rabbit anti-IL-1β (1:300; Abcam, Cambridge, MA, USA). Secondary antisera were Alexa Fluor 488 of donkey anti-rat (1:1000; dilution) and Alexa Fluor 594 of donkey anti-rabbit (1:1000 dilution). Immunofluorescence was performed according to the following procedures. Briefly, the frozen sections were first washed with PBS three times (5 mins/time) and blocked with 1% bovine serum albumin (Sigma) solution at room temperature for 1 h. The sections were incubated for 2 h with primary antibodies at 25 °C. After washing with PBST four times (10 mins/time), sections were then incubated with fluorochrome-conjugated secondary antibodies for 1 h. Each of the above steps was followed by four 10-min rinses in PBST. Cell nuclei were counterstained with DAPI. Images were captured using a confocal microscope (Olympus 1X81 microscope). Positive staining of the sections was confirmed by comparing with the negative Ctrls.