Rabbit anti h3k4me3

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%).

C. elegans dauer larvae and post-dauer adult gonads were dissected, fixed, and stained as described elsewhere [45 (link)]. Extruded gonads were incubated with rabbit anti-H3K4me3 (1:500 dilution, Abcam, ab8580) or rabbit anti-H3K9me3 (1:500 dilution, Cell Signalling Technology, 9754S). Secondary antibodies were Alexa-Fluor-488–coupled goat anti-rabbit (1:500; Life Technologies, Carlsbad, CA, USA). Gonads were counterstained with DAPI (0.1 μg/mL dilution, Roche Diagnostics, 10236276001). Microscopy was performed as described elsewhere [10 (link),46 (link)]. Ratios for the fluorescence intensity across the germ line were determined using ImageJ. The ratio was calculated by dividing the immunofluorescence signal of either anti-H3K4me3 or anti-H3K9me3 by the fluorescence signal of DAPI per nucleus.

Paramecium cells were permeabilized for 20 min in PHEM buffer with 1% Triton X-100, fixed in 2% paraformaldehyde for 10 min and denatured with 0.1-M HCl for 5 min. The cells were then washed with phosphate buffered saline (PBS) for 5 min and blocked for 1 h in TBSTEM buffer with 3% BSA. After blocking the cells were incubated with the primary antibody (rabbit anti-H3K27me3, 1:100, Millipore, cat: 07-449, lot: 2275589 or rabbit anti-H3K4me3, 1:500, Abcam, cat: ab8580, lot: GR144288-1) diluted in TBSTEM buffer with 3% BSA + 0.1% Triton X-100 for 1 h and then washed three times for 10 min with PBS. This was followed by secondary antibody incubation (1:500 goat anti-rabbit conjugated with AlexaFluor 568 or 1:200 goat anti-rabbit conjugated with AlexaFluor 488) at 37°C for 1.5 h. The cells were again washed three times for 10 min with PBS and then stained with DAPI (1 mg/ml) for 5 min. The cells were then washed once with PBS and mounted with Prolong^® Gold Antifade mounting medium (Life Technologies) on a glass slip sealed with a coverslip. Imaging was performed with Olympus FLUOVIEW FV1000 confocal laser scanning microscope and images were processed with Imaris software (Bitplane).