Mca2060

Coronal brain sections (thickness 50 μm) were obtained from the fixed brain immersed in PFA at least 3 d using a vibratome (VT 1000S, Leica, Germany) and rinsed with 0.1 M phosphate buffer saline for 3 times. For NeuN (1:500; MAB377, Millipore, Darmstadt, Germany) and DCX (1:500; sc-271390, Santa Cruz Biotechnology, Shanghai, China) with BrdU (1:500; MCA2060, Serotec, Düsseldorf, Germany) double-immunostaining, the sections were pretreated with 2 M HCl for 30 min at 37 ℃ and subsequently neutralized by 0.1 M borate buffer (pH 8.5), followed by rinsing with 0.1 M phosphate buffer saline for 3 times. The sections were incubated with primary antibodies which were diluted in a blocking solution at 4 °C overnight. Then sections were incubated with secondary antibodies as follows: Alexa Fluor 568 donkey anti-mouse IgG (1:500; A10037, Invitrogen), Alexa Fluor 647 donkey anti-rat IgG (1:500; A48272, Invitrogen), and Fluor 568 donkey anti-goat IgG (1:500; A11057, Invitrogen). Images were acquired using a laser-scanning confocal microscope (OLYMPUS FV3000, Japan). Stereological counts of the total number of positive cells were performed by an investigator blind to treatment by using ImageJ software (https://imagej.nih.gov/ij/index.html).

5-Bromo-2′-deoxyuridine (BrdU, B5002, Sigma-Aldrich, St. Louis, MO) was dissolved in sterile DPBS (10 mg/ml) and given i.p. at 80 mg/kg. At different time-points (see Results) animals were deeply anesthetized with an overdose of ketamine/xylazine, and perfused with 4% PFA. Vibratome sections were used for immunohistochemical detection of BrdU with anti-BrdU mouse monoclonal (1:330, B8434, Sigma-Aldrich) and rat monoclonal (1:100, MCA2060, AbDSerotec) antibodies after DNA denaturation: 30 min treatment with 2 M HCl (RT) followed with 20 min treatment with 0.1 M sodium borate buffer (pH 8.5) at RT.

Mice were intraperitoneally injected with BrdU (50 mg/kg) once every 12 h for 1 week. PFA-fixed brains were dissected, post-fixed and dehydrated as described above. Forty-micrometers cryosections were collected and treated with 1 M HCl for 30 min at 45 °C for DNA denaturation. After rinsing in PBS (three times, 5 min each), sections were incubated in a mixture of 0.3% Triton X-100 and 5% horse serum in PBS for 1 h, incubated in rat-anti BrdU antibody (AbD Serotec, MCA2060, 1:300) at 4 °C overnight, rinsed in PBS (three times, 5 min each) and amplified by Alexa Fluor-488 donkey anti-rat antibody (Life Technologies, A21208, 1:600). Sections were mounted with Fluoromount containing DRAQ-5 (1:1000).

For BrdU detection, see “Immunohistochemistry” Section above. Concerning IdU and CldU immunohistochemistry, sections were washed five times in TBS (Tris-buffered saline), pretreated with 2N HCl for 10 min, then washed five times in TBS. Antibodies were incubated in TBS containing 5% normal donkey serum and 0.25% Triton X-100 (Vega and Peterson, 2005 (link)).
Halogenated thymidine analogs were detected as follows: for IdU using the mouse monoclonal anti-BrdU (BD Biosciences, San Jose, CA, USA; BD 44; 1:500); for CldU using the rat monoclonal anti-BrdU (AbD Serotec, Raleigh, NC, USA; MCA2060; 1:250). Antibody specificity was analyzed in mice that were injected with either IdU or CldU alone. Secondary antibodies used to visualize the antigens were a donkey anti-rat antiserum conjugated to TRITC (CldU) and a donkey anti-mouse antiserum conjugated to Cy2 (IdU), all from Jackson ImmunoResearch (West Grove, PA, USA).

For IP-Western blots, 350μg of protein from lysates was used, and MYC was pulled down with the N262 antibody (sc-764; 2μg/sample). Western blot analysis was performed as described previously [21 (link)]. Immunoblots were visualized using the Odyssey IR imager (LI-COR) that can detect both Fluor 680 and IRDye 800 secondary antibodies (1:10000). Additional antibodies used for western blot include: monoclonal pS62-MYC (1:500, Abcam), Total MYC (Y69) (1:1000, ab32072, Abcam), B56α (1:500, ab72028, Abcam), β-Actin (1:10000, A5441, Sigma) and GAPDH (1:10000, AM4300, Ambion).
Mouse tissues were collected and fixed in 10% formalin-neutral buffer. Paraffin embedding and Hematoxylin and Eosin (H&E) staining were performed at OHSU Histopathology Core. Antibodies used for IF or IHC: pS62-MYC antibody (1:100) we developed as previously described [11 (link)] and pS62-MYC (1:500, ab185656, Abcam), Ki67 antibody (1:1000, Novocastra, NCL-Ki67-MM1), anti-BrdU (1:200, MCA2060, AbD serotec), Cdk4 (1:50, sc-260, Santa Cruz Biotechnology), and CD3 (1:100, Dako). IHC images were scanned and quantified by Aperio ImageScope 11.2.0.780 (Aperio Technologies). Antibodies used for the Flow Cytometry assay was described previously [35 (link)].

All antibodies were diluted in PBS 0.1 M containing 10% goat serum. The primary antibodies for immunofluorescence used in this study were monoclonal rat anti-BrdU MCA2060 (Abd Serotec, Bio-Rad Laboratories, Inc., 1 : 400.) monoclonal mouse and anti-nestin MAB353 clone rat 401 (Millipore, Merck KGaA, Darmstadt, Germany 1 : 100). Secondary antibodies were as follows: Alexa Fluor 488 for BrdU label and Alexa Fluor 594 for nestin label (Invitrogen™, Thermo Fisher Scientific Inc., 1 : 1000). For Western Blot, we used the following: anti-bcl2 rabbit polyclonal (Novus Biologicals, LLC, USA, 1 : 1000), anti-Bcl-xL mouse monoclonal (Sigma-Aldrich Co., St. Louis, MO, USA, 1 : 1000), and anti-β-actin (Santa Cruz Biotechnology, USA, 1 : 2500). Secondary antibodies included biotinylated anti-rabbit and anti-mouse (Vector Laboratories Ltd., Southfield, MI, 1 : 1000).