Mouse anti h3k27me3 antibody

Immuno-FISH was performed as described [65 (link)] with some modification. All ES and EB culture were dispersed by Accutase (Innovative Cell Technologies) and cyto-spinned onto slides at 1,500 rpm for 10 min. After Triton X-100 permeabilization in the cytoskeleton (CSK) buffer, the cells were fixed by 4% paraformaldehyde for 10 min at room temperature. An immunofluorescence assay using a mouse anti-H3K27me3 antibody (Active Motif, #61017) and an Alexa Fluor 555-labeled secondary antibody against mouse IgG (Life Technologies) was followed by 4% paraformaldehyde fixation for 10 min. RNA FISH was carried out with a 25 nM oligonucleotide probe cocktail, which hybridized from exon 1 to 6 of Xist RNA, in the hybridization buffer (10% formamide, 2x SSC, 2 mg/ml BSA, 10% dextran sulfate) at 37°C overnight. The next day, the slides were washed in wash buffer (10% formamide and 2x SSC), wash buffer with 5 ng/ml DAPI, and 2x SSC for 5 min each at 37°C. They were then treated with an antifade solution.
Immunofluorescence was carried out as described previously [35 (link)] using human HEK293T cells. Anti-human hnRNP-U (Santa Cruz, sc-32315) and anti-histone macroH2A (Upstate, 07–219) antibodies were used. The siRNA-mediated gene knockdown for human hnRNP U was performed with Silencer Select Pre-designed siRNA (siRNA ID: s6743, Ambion).

Immunostaining, image acquisition, and analyses were performed as described previously (Inoue et al, 2018 (link)). Embryos were fixed with 3.7% paraformaldehyde and 0.2% Triton X‐100 for 20 min at room temperature. After washing 4 times with PBS containing 1% BSA, samples were incubated with the rabbit anti‐H3K27ac antibody (1/500; Millipore, 07–360) and/or the mouse anti‐H3K27me3 antibody (1/500; Active Motif, 61017) as primary antibodies at 4°C overnight. Then, samples were washed with PBS/BSA and incubated with Alexa flour 568 donkey anti‐rabbit IgG and/or Alexa flour 488 donkey anti‐mouse IgG (1/200; Life Technologies) as secondary antibodies for 1 h at room temperature. After washing with PBS/BSA, samples were mounted on a slide containing VectaShield Antifade Mounting Medium with DAPI (Vector Laboratories). Fluorescent intensities were quantified with Zeiss Zen software.