Anti dnmt1 antibody

Immunohistochemistry for DNMT1 done on formalin-fixed, paraffin-embedded tissue using anti-DNMT1 antibody from Abcam (Cambridge, MA) overnight at a dilution of 1:500 and were stained using automated staining platform (Ventana). Envision + polymer (ready to use; Dako) was used as a secondary antibody. Color was developed with 3,3′-diaminobenzidine (DAB) and instant hematoxylin (Shandon) was used for counterstaining. The DNMT1 level was evaluated and verified by two qualified pathologists, who scored both the proportion of positive cells as well as the intensity of DNMT1 expression in both cancer cells and their stromal fibroblasts.
For CD31, the number of CD31-positive vessels was counted in five different highest fields of microvessel density (40x objective lens and 10x ocular lens). CD31[P2B1] (ab24590) was purchased from Abcam.

ChIP analysis was performed according to the instructions provided with the ChIP assay kit (Millipore, Billerica, MA, USA). In brief, CD14⁺ monocytes were fixed for 10 min at RT with 1% formaldehyde. Glycine was subsequently added to a final concentration of 0.125 M to quench the formaldehyde. Cells were pelleted, washed once with ice-cold PBS, and lysed with SDS buffer. Lysates were pelleted, resuspended, and sonicated to reduce DNA to fragments of 200 to 1000 base pairs. Chromatin was precipitated with protein A agarose beads for 1 h and then incubated with an anti-RFX1 antibody (Santa Cruz), anti-DNMT1 antibody (Abcam), anti-HDAC1 antibody (Abcam), anti-SUV39H1 antibody (Abcam), histone H3ac (pan-acetyl) antibody (Active motif), histone H4ac (pan-acetyl) antibody (Active motif), and histone H3 trimethyl Lys9 antibody (Active motif) or normal IgG (Millipore, Darmstadt, Germany) overnight. The immunocomplexes were further precipitated with protein A agarose beads, washed, and eluted in 100 mL of TE with 0.5% SDS and 200 mg/mL proteinase K. Precipitated DNA was further purified with phenol/chloroform extraction and ethanol. The relative enrichment level was quantified using qPCR and calculated relative to the respective input DNA [33 (link)]. The primers used are shown in Additional file 1: Table S1.

Frozen sections harvested from cows in lactation periods with high and low milk qualities were fixed in acetone at 4°C for 10 min. The tissue sections on slides were washed with phosphate buffered saline (PBS) containing 5% Tween-20 (PBST) and incubated with 5% BSA at 37°C for 60 min. The slides were then incubated with anti-DNMT1 antibody (1∶200, Abcam, Cambridge, MA, USA) in a humid chamber at 4°C overnight, rinsed three times with PBST and incubated for 60 min with TRITC-conjugated goat anti-rabbit IgG (1∶200, Zhongshan-Bio, Beijing, China). The slides were then washed three times with PBST and incubated in 1 µg/ml propidium iodide (PI) (Roche, Florence, Carolina, USA) for 10 min. Finally, 100 µL Antifade Mounting Medium (Beyotime, China) was added. We viewed the slides under a laser scanning confocal microscope (Leica, Germany). Image-Pro Plus (IPP) 6.0 software was used for detecting the mean density of the DNMT1 expression. Three sections from each group were used to quantify the DNMT1 protein.