Mouse anti bcl 2

Frozen sections were washed with distilled water, incubated in 3% hydrogen peroxide solution for 15 min at room temperature, washed with distilled water again, and blocked with 1% bovine serum albumin for 20 min at room temperature. The sections were incubated with primary mouse anti-chicken Bu-1 (Santa Cruz, Texas, USA, 1:100), rabbit anti-Bax (Santa Cruz, Texas, USA, 1:200), and mouse anti-Bcl-2 (BD Biosciences, California, USA, 1:200) antibodies overnight at 4°C. After rinsing with PBS, the sections were treated with goat anti-mouse and anti-rabbit immunoglobulin G (IgG) conjugated to horseradish peroxidase (Zhongshan Golden Bridge Biotechnology, Beijing, China) at room temperature for 1 h. After treating the sections with chromogen diaminobenzidine (Zhongshan Golden Bridge Biotechnology, Beijing, China) for 10 min at room temperature in the dark, the sections were counterstained with hematoxylin. The primary antibodies were replaced with PBS in the negative controls. The densities of Bu-1 in the bursa were identified in 10 high-power lymph nodes using Image-Pro plus 6.0, and the means were calculated. The intensities of Bax and Bcl-2 in 10 high-power fields for each sample were determined using Image-Pro plus 6.0, and the means were calculated. Sampling of the sections was unbiased; all the samples were coded and the examinations were performed by a single investigator.

Western blot analysis was performed as previously described^{43 (link)} using the following antibodies: mouse anti-NOXA, rat anti-BMF, rabbit anti-MCL-1 (Enzo Life Science, Farmingdale, NY, USA), mouse anti-BCL-2, rabbit anti-BAK (BD Biosciences), rabbit anti-caspase-3, rabbit anti-caspase-9, rabbit anti-BIM, mouse anti-PARP, rabbit-anti PUMA, rabbit-anti BCL-x_L (Cell Signaling, Beverly, MA, USA), mouse anti-GAPDH (HyTest, Turku, Finland), rabbit anti-H3K4me2 (Diagenode, Liège, Belgium), rabbit anti-acetylated histone H3 (Merck Millipore, Darmstadt, Germany) and mouse anti-histone H3 (Abcam) or mouse anti-β-Actin (Sigma, Germany). Goat anti-mouse, goat anti-rabbit and goat anti-rat IgG conjugated to horseradish peroxidase (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and enhanced chemiluminescence (Amersham Biosciences, Freiburg, Germany) or infrared dye-labeled secondary antibodies and infrared imaging (Odysee Imaging System, LI-COR Biosciences, Bad Homburg, Germany) were used for detection. For detection of histone modifications cells were lysed using RIPA buffer supplemented with Pierce Nuclease (Thermo Fisher, Waltham, MA, USA). Representative blots of at least two independent experiments are shown.