Biotinylated secondary donkey anti rabbit antibody

IHC staining was performed as described in our recent studies (Shen et al., 2022a (link); Yao et al., 2022 (link)). Briefly, following deparaffinization and antigen retrieval, FFPE slides were blocked in phosphate-buffered saline (PBS) containing 4% donkey serum and 0.2% Triton X-100 for 1 h at room temperature. Then FFPE slides were incubated with primary antibody to ACSL4 at a dilution of 1:250 (Abcam, MA) overnight at 4°C; the sections were washed with PBS three times and incubated with biotinylated secondary donkey anti-rabbit antibody (Vector Laboratories, CA) in PBS with 0.2% Triton X-100 at a dilution of 1:500 for 1 h. Sections were washed again, and the secondary antibodies were detected by use of a DAB detection kit (Vector Laboratories), followed by counterstaining in 50% hematoxylin for 2 min.
ACSL4 staining in IHC was quantified with use of ImageJ, an image processing program developed at National Institutes of Health (Schneider et al., 2012 (link)). The mean of percentage of staining area by ACSL4 protein in the chorionic villi tissues was acquired from three random fields. We set the overall scores from 0 to nine based on the percentage staining area of <0.1%, ≥0.1 and <1%, ≥1 and <2%, ≥2 and <3%, ≥3% and <4%, ≥4 and <5%, ≥5 and <6%, ≥6 and <7%, ≥7 and <8%, and ≥9%, respectively.

After calcium imaging, explants (6–11 d) were fixed for 30 min with 0.1 m PBS, pH 7.4, containing 4% formaldehyde at room temperature. After a few washes in PBS, explants were incubated in a blocking solution (10% normal horse serum plus 0.3% Triton X-100) for 1 h, and washed several times in PBS. The explants were incubated at 4°C overnight in the primary antibody (rbGnRH; Wray et al., 1989b (link); Table 4). The next day, explants were washed in PBS, incubated for 1 h with biotinylated secondary donkey anti-rabbit antibody (1:500 in PBS/0.3% Triton X-100; Vector Laboratories), washed in PBS, and processed for avidin–biotin horseradish peroxidase/3,3´-diaminobenzidine (Fig. 1A).

After calcium imaging, explants (6–11 d) were fixed for 30 min with 0.1 m PBS pH 7.4 containing 4% formaldehyde at room temperature. After a few washes in PBS, explants were incubated in a blocking solution (10% normal horse serum + 0.3% Triton X-100 + 0.1% NaAzide) for 1 h and washed several times in PBS. The explants were incubated at 4°C overnight in the primary antibody (in PBS with 1% BSA + 0.1% NaAzide; SW-1, Wray et al., 1989 (link); Table 2). The next day, explants were washed in PBS, incubated for 1 h with biotinylated secondary donkey anti-rabbit antibody (1:500 in PBS/0.3% Triton X-100; Vector Laboratories, Inc), washed in PBS, and processed for avidin-biotin horseradish peroxidase/3,3′-diaminobenzidine (Fig. 1A).