Polhrp 100

To explore the histone modification levels in placentas from natural conception and ARTs. Global H3K4me3, H3K9ac, and H3K27ac were compared via IHC on placental tissues. After pre-treatment of formalin-fixed, paraffin-embedded sections, placental tissues were incubated with primary antibodies for 16 h at 4 °C. The primary antibodies included rabbit anti-H3K4me3 (1:100; Abcam, Cambridge, MA, USA, Cat# ab8580; RRID: AB_306649), anti-H3K9ac (1:200, Abcam Cat# ab32129, RRID: AB_732920), anti-H3K27ac (1:2,000; Abcam, Cat# ab177178; RRID: AB_2828007), mouse anti- Polr2A (1:500, OriGene, Rockville, MD, USA, Cat# CF810050), rabbit anti- KDM5A (1:300, Thermo Fisher Scientific, Waltham, MA, USA, Cat# PA5-50741; RRID: AB_2636193). For detection, a horseradish peroxidase-coupled anti-mouse/rabbit polymer system (Zytomed Systems, Berlin, Germany, Cat# POLHRP-100) was used with DAB (Dako, Glostrup, Denmark, Cat# K3468) as the chromogen. The expression of primary antibodies was evaluated by two independent, blinded observers using semi-quantitative IRS [57 (link)]. IRS (range of 0 to 12) was obtained by multiplying the score of intensity (0 = no; 1 = weak; 2 = moderate; or 3 = strong staining) and that of the extent of positive cells (0 = none; 1 = 1–10%; 2 = 11–50%; 3 = 51–80%; 4 = 81–100%).

ICC was used to study the global levels of H3K4me3 after siRNA transfection. Cells were fixed on slides and incubated with rabbit anti-H3K4me3 antibodies (1:500; Abcam, Cat# ab8580; RRID: AB_306649) for 16 h at 4 °C. HRP-coupled anti-mouse/rabbit polymer system (Zytomed Systems, Cat# POLHRP-100) was used for detection, with DAB (Dako, Cat# K3468) as the chromogen. The levels of H3K4me3 were evaluated by two independent, blinded observers using IRS [57 (link)].