Sea block blocking solution

Cells were fixed with 4% paraformaldehyde (PFA) and then washed with PBS. For 4°C experiments, cells were maintained at 4°C for 10 minutes before treatments, and maintained at that temperature for an additional 30 minutes before fixation. Nonspecific binding was prevented using Sea Block blocking solution, for 1 hour at RT (Thermo Fisher Scientific). Cells were kept overnight at 4°C in a primary antibody solution and incubated for 1 hour at RT with the corresponding secondary antibody. Antibodies were used as listed: rabbit anti‐Ago‐2 (1:100) (Cell Signaling Technology Inc); rabbit anti‐CD8 (1:50) (Santa Cruz Biotechnology Inc); Alexa Fluor 594 goat anti‐rabbit (1:200) (Molecular Probes, OR). For nuclear labeling, cell preparations were stained with Hoechst‐33342 (2 μg/mL) (Sigma) and mounted in Dako Fluorescence Mounting Medium (Dako North America Inc). Fluorescent images were acquired using an LSM 510 confocal microscope with a 40× objective (Carl Zeiss Inc).

Uterine tissue was obtained from pregnant and unfertilized mice, placed in optimal cutting temperature (Beijing Solarbio Science & Technology Co., Ltd., Beijing) medium and frozen. Frozen tissue sections were rehydrated and treated for 30 min with 1X PBS/0.25% Triton X-100 solution, and subsequently rinsed in PBS twice for 5 min. Tissue sections were then incubated for 40 min in undiluted Sea Block Blocking Solution (Thermo Fisher Scientific, Inc.), rinsed in PBS and incubated in undiluted Fc Receptor Blocker solution (Innovex Biosciences, Inc., Richmond, CA, USA) for 30 min. After rinsing in PBS, tissue sections were incubated overnight at 4°C with a primary antibody against TRPC3 (1:300; no. ab51560; Abcam). Sections were then washed in PBS three times for 10 min and then incubated with the suitable fluorochrome-labeled secondary antibodies (goat anti-rabbit Alexa Fluor 488; cat. no. ab150077; Invitrogen/Molecular Probes; Thermo Fisher Scientific, Inc.). Chromatin was counterstained with Hoechst (10 mg/ml; Sigma-Aldrich; Merck KGaA) diluted in H₂O (1:10,000). Specimens were examined by confocal microscopy with an Olympus FV-1000 laser scanning confocal microscope (HeNe/Ar) with a 60× water immersion objective, and images were processed with the FluoView software (Olympus Corporation). Selected representative fields are presented.