3.3 diaminobenzidine substrate dab

Immunohistochemistry was conducted as previously described [47 (link)]. Tissue was cryosectioned at 40 μm (Leica Microsystems). Free-floating sections were incubated in mouse anti-NeuN (1:500; Merck Millipore) for 72 h followed by overnight incubation in biotin-labeled chicken anti-mouse secondary antibody (1:250; Invitrogen). Immunolabeling was detected using HRP-labeled avidin-biotin complex and 3.3′-Diaminobenzidine substrate (DAB; Vector Laboratories).

Mice brain tissues were fixed in 10% neutral buffered formalin and embedded in paraffin blocks. 4-μm-thick brain sections were cut and used for immunohistochemical (IHC) analysis. After deparafinization and rehydration, heat-mediated antigen retrieval and blocking, sections were incubated with primary antibodies to REST (Cat# IHC-00141; Bethyl, Montgomery, TX), CD31 (Cat# ab28364; Abcam, Cambridge, MA), VEGFR2 (Cat# 2479; Cell Signaling Technology, Danvers, MA), Ki67 (Cat# AB9260; Millipore, Billerica, MA), and Gremlin-1 (Cat# PA5-13123; Thermo-Fisher Scientific, Waltham, MA) at 4°C overnight. After washing, sections were incubated with secondary antibody conjugated to horse radish peroxidase (Cat# 111-035-003 and Cat# 115-035-003; Jackson ImmunoResearch Labs, West Grove, PA) for 2 h at room temperature. All incubations were performed under humidified conditions. After washing, slides were developed using 3,3′-diaminobenzidine substrate (DAB; Cat# Vector Laboratories, Burlingame, CA), counterstained with hematoxylin, dehydrated, mounted and visualized under a microscope (Nikon ECLIPSE E200; Melville, NY). Images were obtained using an Olympus SC100 camera (Waltham, MA) attached to the microscope. Images were processed using CellSens Entry microscopy imaging software (Olympus Life Sciences, Waltham, MA).