4.5. Immunohistochemistry for NLRP3 and Double Immunofluorescence for NLRP3/Iba1 or ASC/NLRP3

Brain samples for histological assessment were collected at one day after ischemic challenge. Mice were anesthetized with a mixture of Zoletil 50^® (10 mg/kg, i.m., Virbac Laboratories, Carros, France) and Rompun^® (3 mg/kg, i.m., Bayer HealthCare LLC, Shawnee Mission, KS, USA), perfused transcardially with ice-cold phosphate-buffered saline, and fixed with paraformaldehyde (PFA). Brains were removed, post-fixed with 4% PFA overnight, immersed in 30% sucrose solution, embedded in Tissue-Tek Optimal Cutting Temperature compound, and frozen on dry ice. These frozen brain samples were cut into 20-µm coronal sections using a cryostat (RD-2230, Roundfin, Liaoning, China).

For NLRP3 immunohistochemistry, brain sections were post-fixed in PFA, exposed to 0.01M sodium citrate at 90–100 °C and 1% H₂O₂, blocked with 1% fetal bovine serum (FBS), and incubated with a mouse anti-NLRP3 primary antibody (1:200, AdipoGen Life Sciences, San Diego, CA, USA) overnight at 4 °C, followed by labeling with a biotinylated secondary antibody (1:200, Santa Cruz Biotechnology, Dallas, TX, USA), for 2 h at room temperature (RT). These sections were further incubated with ABC reagent (1:100, Vector Laboratories, Burlingame, CA, USA). Signals were developed with a DAB kit (Dako, Santa Clara, CA, USA), rinsed with water, dehydrated with alcohol and xylene, and mounted with an Entellan media (Merck, Darmstadt, Germany).

For NLRP3/Iba1 double immunofluorescence, brain sections after blocking with 1% FBS were incubated with mouse anti-NLRP3 (1:100) and rabbit anti-Iba1 (1:500, Wako Pure Chemicals, Osaka, Japan) primary antibodies overnight at 4 °C. In case of ASC/NLRP3 double immunofluorescence, rabbit anti-ASC (1:200, AdipoGen Life Sciences) and mouse anti-NLRP3 (1:100) primary antibodies were used. Sections were then labeled with AF488- and Cy3-conjugated secondary antibodies (1:1000, Jackson ImmunoResearch West Grove, PA, USA) for 2 h at RT, followed by counterstaining with 4’,6-diamidino-2-phenylindole (DAPI) (Carl Roth, Karlsruhe, Germany). Labeled sections were mounted with VECTASHIELD^® (Vector Laboratories).

Bright-field or fluorescence images were photographed with a microscope equipped with a DP72 camera (BX53T, Olympus, Tokyo, Japan) or with a confocal microscope (Eclipse A1 Plus, Nikon, Tokyo, Japan). All representative images were prepared using Adobe Photoshop Elements 8. For quantification, three different photos (600 µm × 600 µm) of each brain region were taken in a blind fashion. The number of immunopositive cells in each photo was manually counted and then converted to the number of immunopositive cells per unit area (mm²). The mean was used for the number of immunopositive cells for the region of a single mouse. In case of ASC/NLRP3 double immunofluorescence, the number of ASC/NLRP3-double immunopositive cells of each photo (200 µm × 200 µm) was manually counted and then used for calculating % of cells with ASC/NLRP3 specks versus total cells (DAPI-positive cells).