Dab peroxidase substrate kit sk 4100

To examine GATA3 and GCM1 expression in placenta, early and term human placental tissue biopsy specimens were fixed in 4% paraformaldehyde, dehydrated, embedded in paraffin, and sectioned at 5 μm. Tissue sections were deparaffinized, rehydrated, and subjected to immunostaining by incubation with GCM1, GATA3 (Santa Cruz Biotechnology, Santa Cruz, CA), and cytokeratin 7 (CK7, EMD Millipore, Billerica, MA) antibodies (Abs) overnight, respectively. The sections were then incubated sequentially with biotinylated secondary Ab (Jackson ImmunoResearch, West Grove, PA) and HRP-conjugated streptavidin (Jackson ImmunoResearch). Antigenic detection was performed using DAB peroxidase substrate kit SK-4100 (Vector Laboratories, Burlingame, CA) and the sections were further counterstained with hematoxylin. Co-localization of GCM1 and GATA3 was performed in BeWo and primary human trophoblast cells by co-staining both factors with GCM1 and GATA3 Abs overnight, and then co-incubated with rhodamine-labeled secondary Ab for GCM1 and Cy2-labeled secondary Ab (Jackson ImmunoResearch) for GATA3. Nuclei were stained by DAPI. Immunofluorescence was examined under a Zeiss laser scanning confocal microscope (LSM510).

Immunohistochemistry analysis was performed as previously described [41 (link)]. Briefly, uterine sections were pre-incubated with 10% normal goat serum in PBS prior to exposure to anti-PGR (SC-538; Santa Cruz Biotechnology, Dallas, TX), anti-ESR1 (SC-543; Santa Cruz Biotechnology, Dallas, TX), anti-AKT (CS-4691; Cell Signaling, Danvers, MA), anti-pAKT (CS-4060; Cell Signaling, Danvers, MA), and anti-Ki67 (BD5506090; BD Biosciences, San Jose, CA) as appropriate primary antibodies. Positive signaling was detected with the DAB Peroxidase Substrate Kit (SK-4100; Vector Laboratories, Burlingame, CA). The H-score was calculated as previously reported [42 (link)]. The overall H-score ranged from 0 to 300.

Free-floating sections were washed in PBS and then treated with 3% H₂O₂ for 30 min, rinsed 3–5 times in PBS, and preincubated in PBS containing 5% normal goat serum. After preincubation, sections were incubated with polyclonal primary antibody anti-TRPV1 (ab31895, Abcam, dilution: 1:500) overnight at 4°C. The next day, sections were rinsed five times in PBS and then incubated for 1–2 h at RT with secondary antibodies including biotinylated goat anti rabbit lgG (BA10001, Vector laboratories, dilution: 1:200). After washing, the sections were incubated with a vectastain ABC complex (PK-4000, Vector laboratories) for 30 min, rinsed again 3–5 times in PBS, developed for 4–6 min in diaminobenzidine (DAB Peroxidase substrate kit SK-4100. Vector Laboratories). After the final rinsing in PBS, the developed sections were wet-mounted on glass slides. Following an air-drying process, sections were cover-slipped with Dako Faramount Aqueous Mounting Medium (S3025, Dako).