Prolong anti fade reagents

HeLa cells were cultured in a six-well plate with 22-mm coverslips. YFP-p300 plasmids were transfected into HeLa cells by Lipofectamine 3000 (Life Technology, L3000-075) according to manufacturer instructions. One day after transfection, cells were fixed by 1% paraformaldehyde and then permeabilized with 0.2% Triton X-100. After blocking with 3% goat serum and 3% BSA, cells were incubated, respectively, with pairs of primary antibodies: anti-GFP (Life Technologies; A-11120; 1:400 dilution) and anti-H3K27me3 (Millipore; 07-449; 1:200 dilution), anti-GFP (Life Technologies; A-11120; 1:400 dilution) and anti-H3K9me3 (Upstate; 07-442; 1:200 dilution), and anti-GFP (Life Technologies; A-11120; 1:400 dilution) and anti-H3K4me3 (Novus; NB21-1023B; 1:200 dilution), for two hours. After washing, cells were incubated with a pair of secondary antibodies: Alexa Fluor 488-labeled goat anti-mouse and Alexa Fluor 568-labeled goat anti-rabbit, for two hours, and then were mounted by using ProLong Antifade reagents (Life Technologies; P7481). Line plots were made using the plot profile feature of ImageJ. Pearson coefficient correlation for each pair of immunofluorescence images was calculated using the Coloc 2 plugin in ImageJ after the selection of the entire cell nuclei as the regions of specific interest.

Whole tissue (popliteal lymph node or hepatic lymph node) was collected and placed in Tissue-Tek optimum cutting temperature compound (OCT) (Thermo Scientific) and frozen in liquid nitrogen. Serial cryostat sections (10μm) were collected using a Leica CM 1850. Sections were then air-dried and fixed in ice-cold 75% acetone/25% ethanol for 5 mins. Sections were rehydrated in PBS for 5 to 10 minutes and blocked using a biotin blocking kit (Vector Laboratories) followed by incubation with 1%(v/v) in PBS of rat and rabbit serum. Staining with appropriate antibodies was done overnight at 4°C followed by secondary staining for 1 hour at room temperature. Coverslips were mounted using ProLong anti-fade reagents (Life Technologies). Images were acquired with a Leica TCS Sp5 Laser Scanning Microscopy with an average grid size of 3x3. Images were taken with a 20x objective at a resolution of 1024x1024. Image post-processing was done using Fiji is Just ImageJ software (1.47v).

RAW 264.7 cells were grown on sterile microscope slides, and treated with NKS3 (50 µM for 2 h) and then LPS (10 ng/ml for 24 h). At the end of the incubation, cells were washed with PBS and slides were fixed in 95% ethanol and rehydrated in 0.1 M PBS (pH 7.4). Slides were blocked in PBS containing 5% fetal calf serum and 0.2% Triton-X100 for 30 min at room temperature before overnight incubation at 4 °C with NF-κB p65 antibody (Cell Signalling, D14E12) (1/100 dilution). After washing, slides were incubated for 2 h at room temperature with rhodamine-conjugated secondary antibodies. Staining specificity was assessed by treating slides in the absence of primary antibodies. After three washings with PBS, a drop of ProLong Antifade reagents containing DAPI (Life technologies) was added onto the slide for the analysis under fluorescent microscope (Zeiss Axioskop).

Wild-type (PGK12.1), Eed^─/─, Ezh2^─/─, HaloTag-Eed/Eed^─/─, and HaloTag-Ezh2/Ezh2^─/─ mES cells and SF8628, 9427, HaloTag-Cbx7/SF8628, and HaloTag-Cbx7/9427 cells were fixed using 2.0% paraformaldehyde. After permeabilizing with 0.2% Triton X-100, we washed the cells with basic blocking buffer (10 mM PBS pH 7.2, 0.1% Triton X-100, and 0.05% Tween 20) and then with blocking buffer (the basic blocking buffer plus 3% goat serum and 3% bovine serum albumin) overnight. Anti-H3K27me3 antibody (9733; Cell Signaling Technology; 1:200 dilutions) or anti-Cbx7 antibody (ab21873; Abcam; 1:250 dilutions) was incubated with the cells for 2 h at room temperature. After washing with the basic blocking buffer, we incubated cells with Alexa Fluor® 488-labeled goat anti–rabbit antibody (A-11008; Life Technologies; 1:1000 dilutions) for 1 h. After incubating with 25 ng per ml hoechst, we washed and mounted the cells on slides with ProLong Antifade reagents (P7481; Life Technologies).