Anti sqstm1 antibody

Uteri were collected from female mice with the indicated genotypes, cut into 3 to 5 mm pieces, and fixed in 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS) overnight at 4 °C. The uterine pieces were washed with PBS and embedded in sucrose solution for several hours until the pieces were completely submerged in the solution. They were flash-frozen in optimal cutting temperature compound and subjected to cryosection (12 μm thickness). The uterine sections were then fixed in 4% PFA-PBS for 20 minutes, washed, and permeabilized with 0.1% Tween-20 in PBS for 20 minutes. The specimens were blocked with 2% bovine serum albumin (BSA) in PBS for 1 hour at 25 °C and then incubated with anti-SQSTM1 antibody (Cell Signaling Technology) in 2% BSA-PBS for 2 hours at 25 °C [2 (link)]. After washing, the sections were incubated with Alexa Fluor 488-conjugated secondary antibodies (Thermo Fisher Scientific) for 40 minutes. The sections were counterstained with TOPRO-3-iodide (Thermo Fisher Scientific). The mounted slides were observed by live confocal imaging using a Zeiss LMS900 confocal microscope.