Anti g9a

Rabbit polyclonal L1 ORF1 antisera were made through immunization of rabbits with recombinant ORF1 protein. The following antibodies were used at the indicated dilutions for IF: anti-G9a (1:50) (A. Tarakhovsky, The Rockefeller University, New York, NY, USA), anti-ORF1 L1 (1:500), mouse monoclonal anti-GLP (R&D systems, Minneapolis, MN, USA, PP-B0422-00) (1:100), anti-IAP Gag (1:500) (B. Cullen, Duke University, Durham, NC, USA), mouse monoclonal anti-γH2AX (Abcam, Cambridge, UK ab26350) (1:500), mouse monoclonal anti-H3K9me2 (Abcam, Cambridge, UK ab1220) (1:100) and rabbit polyclonal anti-Plzf (Santa Cruz, Dallas, TX, USA sc-22839) (1:100). Immunofluorescence and histology were performed as described [3 (link)]. Quantification of L1 Orf1 and IAP signal from at least 20 to 40 cells was performed using Fiji software (http://fiji.sc/Fiji).

Embryos were dissected from the decidua and Reichert’s membrane and treated as previously described (Nichols et al., 2009 (link)). Primary antibodies used are as follows: anti-H3K9me2 (Millipore, CA, 17–648), anti-H3K9me2 (Abcam, UK, ab1220), anti-GFP (Nacalai tesque, Japan, GF090R), anti-G9a (R&D Systems, MN, A8620A), anti-GLP (R&D Systems, MN, PP-B0422-00), anti-cleaved Caspase 3 (Abcam, UK, ab32042), anti-Ki67 (BD Bioscience, NJ, 550609), anti-NANOG (Cosmobio, Japan, REC-RCAB002P-F), anti-AP2γ (Santa Cruz, CA, sc-9877). All imaging was performed using SP5 or SP7 confocal microscope (Leica, Germany).

The study was approved by the National Taiwan University Hospital Ethics Committee. Paraffin-embedded tumor blocks were dewaxed and pretreated in citric acid buffer solution (pH 6.0) (for ki67 staining) or boric acid solution (for G9a staining) by microwave boiling for 15 min to retrieve the antigens. Sections were then incubated with 1% H₂O₂ in methanol for 15 min at room temperature to block endogenous peroxidase; they were then blocked with 3% bovine serum albumin for 1 h at room temperature. Staining was performed at 4°C overnight with the indicated antibodies, anti-G9a (R&D Systems, Minneapolis, MN, 1:100) and Ki-67 (GeneTex, 1:100). Sections were incubated with immunoglobulin G (IgG)-biotinylated secondary antibodies (BioGenex) for 40 min then incubated with streptavidin peroxidase (BioGenex) for 30 min at room temperature. Visualization was achieved using 0.03% diaminobenzidine tetrahydrochloride substrate for 3 min, and then sections were counterstained with Mayer’s hematoxylin and mounted. We classified staining intensity having as low (negative and focal expression less than 20% of tumor cells) and high (diffuse expression in more than 20% of tumor cells) expression.