Rabbit igg isotype

ChIP extract preparation followed a previously described method with minor changes [35 (link)]. Briefly, target cells were seeded on 150 mm dishes and cultured until 90% confluence. Cells were fixed with 1% (w/v) paraformaldehyde prior to cross-linking with DTBP. Rabbit polyclonal antibodies raised against BCL6 (SC-858; Santa Cruz Biotechnology, Dallas, TX, USA) and NAC1 (SC-98638; Santa Cruz Biotechnology), or a rabbit IgG isotype (Abcam, Cambridge, MA, USA) were used for immunoprecipitations. The precipitated DNA was used for quantitative or semi-quantitative PCR. The primers used are summarized in Table 1.

Primary antibodies were as follows: mouse anti-a2V (Covance, Denver, PA); rabbit anti-GAPDH (Cell signaling, Danvers, MA); rat anti-F4/80, rabbit anti-LC3B, rabbit anti-LAMP-1, rat anti-LAMP-2, rabbit anti-ATG3, rabbit anti-ATG4, rabbit anti-ATG7, rabbit anti-NF-κB p65 (all from Abcam). Secondary antibodies were as follows: goat anti-rabbit IgG-FITC, -mouse IgG AF-594, -rabbit IgG AF-594 (Invitrogen), rabbit anti-rat IgG-FITC (Abcam), goat anti-rabbit, -rat, -mouse IgG-HRP (Santa Cruz Biotechnology), donkey anti-rabbit IRDye-800CW (LI-COR Bioscience, Lincoln, NE) and EnVision + dual link System-Horseradish peroxidase (HRP) (Dako). Isotype control antibodies were as follows: control mouse IgG (R&D Systems); rat IgG isotype, mouse IgG isotype and rabbit IgG isotype (Abcam).

For IHC, paraffin sections (5 µm) of the EMS-like lesions and uterine endometrium were dehydrated in graded ethanol and then were incubated with hydrogen peroxide and 1% bovine serum albumin/TBS to block endogenous peroxidase. The samples were then incubated with rabbit anti-human Ki-67 (1:500; Abcam, USA), Beclin-1 (1:200; Abcam) and LC3B (1:500; Abcam) or rabbit IgG isotype (Abcam) overnight at 4 °C in a humid chamber. After washing three times with TBS, the sections were overlaid with peroxidase-conjugated goat anti-rabbit IgG, and the reaction was developed with 3,3-diaminobenzidine (DAB) and counterstained with hematoxylin.
For IF, according to a previous procedure^{7 (link)}, EMS-like lesions and uterine endometrium tissues were incubated with anti-goat ERα, anti-goat ERβ, anti-goat PR antibody (1:100, Abcam) in PBS at 4 °C overnight. The slides were then incubated with Alexa Fluor 488-conjugated donkey anti-goat secondary antibody (1:500; Abcam). The nuclei were then stained with 4′,6-diamidino-2-phenylindole (DAPI; Beyotime, China). Images were captured with a confocal microscope (Leica, Germany).