Alexa 488 or alexa 594labeled secondary antibodies

Frozen sections of the mice epidermis were fixed for 10 minutes in acetone at −20°C. Paraffin section deparaffinized in xylene and rehydrated using graded ethanol. Cultured cells were fixed in cold methanol 5 minutes and then in acetone 15 s at 4°C. Antigen retrieval was performed using unmasking solution (Vector Laboratories) or 0.5% Triton X-100 in humidified chamber. Samples were blocked with 0.5%nonfat dry milk and 10% horse serum in PBST 1hour at RT. Primary antibodies were incubated overnight at 4°C. After washing three times with PBST, Alexa 488 or Alexa 594labeled secondary antibodies (Invitrogen) were applied for 30 minutes at RT, followed by another wash and mounted with Prolong Gold antifade reagent with DAPI (Invitrogen).Images were taken using an Olympus BX43.

Frozen slides with mouse kidney tissue were fixed in acetone, blocked, then incubated with primary antibodies, including anti-podocin antibody (1:100; Sigma-Aldrich, St. Louis, MO), anti-desmin antibody (1:100; Invitrogen, Carlsbad, CA), anti-NLRP3 antibody (1:50; Abcam Biotechnology, Cambridge, MA), anti-ASC antibody (1:50; Santa Cruz Biotechnology, Dallas, TX), and anti-CD8 antibody (1:100; Abcam Biotechnology, Cambridge, UK), overnight at 4 °C. Immunofluorescent staining was accomplished by incubating slides with Alexa-488- or Alexa-594-labeled secondary antibodies (Invitrogen, Carlsbad, CA) for 1 h at room temperature [47 (link)]. Slides were washed, mounted, and observed by a confocal laser scanning microscope (FluoView FV1000, Olympus, Tokyo, Japan). Image Pro Plus 6.0 (Media Cybernetics, Bethesda, MD) was used to analyze colocalization which was expressed as Pearson correlation coefficient (PCC).