Anti nlrp3

WT and AnxA1^-/- nigericin-stimulated neutrophils were processed for transmission electron microscopy analysis as previously described [13 (link)]. To detect NLRP3, ultrathin sections (~90 nm) from neutrophils were submitted for immunocytochemistry, using a rabbit polyclonal antibody anti-NLRP3 1:200 (Cusabio, Houston, TX, USA) followed by a goat anti-rabbit IgG antibody (1:50) conjugated to 15 nm colloidal gold (British Biocell, Cardiff, UK). Ultrathin sections were stained with uranyl acetate and lead citrate and examined using a ZEISS EM900 electron microscope (Carl Zeiss, Oberkochen, Germany).

WT and AnxA1^-/- nigericin-stimulated macrophages were fixed in 4% paraformaldehyde and 0.5% glutaraldehyde, 0.1% sodium cacodylate buffer (pH 7.4) for 24 h at 4 °C. Samples were washed in sodium cacodylate, dehydrated through a graded methanol series, and embedded in LR Gold (Sigma Aldrich Corp., St. Louis, MO, USA).
To detect AnxA1 and NLRP3, ultrathin macrophage sections (~90 nm) were submitted for immunocytochemistry, as previously described [19 (link)]. To detect the proteins, the sheep polyclonal antibody anti-AnxA1 (1:200) and rabbit polyclonal antibody anti-NLRP3 (1:200; Cusabio, Houston, TX, USA), following a donkey anti-sheep IgG and goat anti-rabbit IgG antibody (1:50) conjugated to 10-nm and 20-nm colloidal gold (British Biocell, Cardiff, UK), respectively, were used. Ultrathin sections were stained with uranyl acetate and lead citrate and examined using a ZEISS EM900 electron microscope (Carl Zeiss, Jena, Germany). Randomly photographed sections of macrophages were analyzed using Axiovision software. The density of immunogold (number of gold particles/µm²) was calculated and reported as the mean ± SEM of 20–40 cells per experimental condition.