Mouse anti pcna

Embryos were dissected, and the brains were fixed in 4% paraformaldehyde (PFA) for 1–3.5 hr. For postnatal stage, brains were fixed with 4%PFA overnight. Following 30% sucrose replacement, fixed brains were embedded in optimum cutting temperature (OCT) compound, and 20 micrometer slices were cut on a cryostat. The antibodies used were, rat anti-GFP (1∶500; nakalai tesk), rabbit anti-GFP (1∶200; IBL), rabbit anti-DsRed (1∶500; Invitrogen), goat anti-Unc5D (1∶100; R&D), rabbit anti-Tbr2 (1∶300; abcam), goat anti-NeuroD1 (1∶100; Santa Cruz), mouse anti-PCNA (1∶100; Cell Signaling), mouse anti-Tuj1 (1∶500; SIGMA), rabbit anti-Tbr1 (1∶100; abcam), mouse anti-RORb (1∶100; PERSEUS PROTEOMICS), rat anti-Ctip2 (1∶300; abcam), goat anti-Brn2 (1∶100; Santa Cruz), and mouse anti-Prdm8 [24] (link). Alexa Fluor-conjugated secondary antibodies (Invitrogen) were also used. EdU labeling (intraperitoneal injection of 12.5 mg/kg EdU) and staining were performed according to manufacturer's instructions (Invitrogen). Stainings were examined with Zeiss LSM 710 or Olympus IX81, and the images were finally processed with Adobe Photoshop.

For the immunohistochemical analysis, sections were incubated with mouse anti-VEGF (1 : 200, Abcam) or mouse anti-HIF-1α (1 : 200, Novus), and then with biotinylated anti-mouse IgG (1 : 800, Santa Cruz) for 1 h at 37°C. The samples were then incubated with an avidin-biotin-peroxidase complex (1 : 100, Vector Laboratories) for 1 h at 37°C. Immunoreactivity was visualized with DAB.
To detect proliferating cerebral microvascular endothelial cells, the sections were incubated with mouse anti-PCNA (1 : 1400, Cell Signaling Technology) and rabbit anti-vWF (1 : 400, Dakon) for 1 h at 37°C. The following secondary antibodies were then used: fluorescein 488-conjugated anti-rabbit antibody (1 : 1000, Jackson ImmunoResearch) and Cy3-conjugated anti-mouse antibody (1 : 1000, Jackson ImmunoResearch). After washing with PBS, the sections were stained with DAPI for 2 min to reveal the location of the nucleus. PCNA⁺/vWF⁺ nuclei close to the hematoma were counted. The data were presented as the number of nuclei per mm² (N/mm²). To determine whether VEGF and HIF-1α were expressed in the endothelial cells , tissue sections were incubated with mouse anti-VEGF (1 : 200, Abcam) or mouse anti-HIF-1a (1 : 100, Novus) with rabbit anti-vWF (1 : 400, Dakon).

RIPA buffer (0.1% SDS, 150 mM NaCl, 1.0% Triton X-100, 10 mM Tris, 5 mM EDTA pH 8.0) with a protease and phosphatase inhibitor cocktail (Roche Applied Science, Indianapolis, IN, USA) was used to obtain the cell lysates. The Bradford assay was used to evaluate the protein concentration in each sample. Proteins (25 μg) were analyzed by SDS-PAGE, and then transferred to a nitrocellulose membrane (Bio-Rad, Hercules, CA, USA). A blocking buffer (Bio-Rad, Hercules, CA, USA) was used to block the membrane that was then incubated overnight with primary antibodies.
Mouse anti-phospho-p38 (Cell Signaling), rabbit anti-phospho-NF-kB (Cell Signaling), mouse anti-PCNA (Cell Signaling) and mouse anti-β-actin (Cell Signaling) were used as primary antibodies.
We used secondary horseradish peroxidase-conjugated antibodies (anti-mouse or anti-rabbit) (The Jackson laboratory, Bar Harbor, ME, USA). A Uvitec Imager (UVItec, Cambridge, UK) was used to visualize the protein bands by using the Clarity ECL chemiluminescence substrate (Bio-Rad) that were then quantified using ImageJ software. Each measure was normalized with respect to β-actin.

Antibodies used for immunohistochemistry and western blot included rabbit anti-CD8(+) (RM-9116-S1, Thermo Fisher Scientific, Cheshire, UK), rabbit anti-CCL5 (ab9679, Abcam, Cambridge, UK), mouse anti-PCNA (2586, Cell Signaling Technology, MA, USA), rabbit anti-GAPDH (2118, Cell Signaling Technology, MA, USA), mouse anti-β-ACTIN (3700, Cell Signaling Technology, MA, USA), rabbit anti-STAT5 (9363, Cell Signaling Technology, MA, USA), rabbit anti-phospho-STAT5 (9314, Cell Signaling Technology, MA, USA) and rabbit anti-CCND1 (2978, Cell Signaling Technology, MA, USA). The reagents used in the in vitro experiment included recombinant human CCL5 (rhCCL5), anti-CCL5 neutralizing antibody and Pimozide were purchased from R&D systems (278-RN, MAB678, 0937 Minneapolis, MN, USA). rhCCL5 was reconstituted in phosphate buffered saline (PBS) and adjusted to a final concentration of 2, 20, 100 ng/ml. For blocking the proliferation effect of rhCCL5 and conditioned media, the anti-CCL5 neutralizing antibody was reconstituted in PBS and adjusted to a final concentration of 2 μg/ml. The STAT5 inhibitor Pimozide was used for interruption assay at a final concentration of 10 μM (dissolved in DMSO). All reagents were used in a low androgen condition to evaluate the proliferation effect and the mechanism dissection of CD8+ T cells or rhCCL5 on BECs.