Brains of E15 embryos were fixed with 4% paraformaldehyde (Sigma-Aldrich, USA) overnight and then embedded in 30% sucrose to provide cryoprotection to the tissue. Coronal sections (10 μm) were obtained with a cryostat (Leica, Germany), permeabilized with 0.1% Triton X-100 (Sigma-Aldrich, USA), and incubated with 3% bovine serum albumin (Sigma-Aldrich, USA). After standard antigenic retrieval, the following primary antibodies were incubated overnight: mouse J2 anti-dsRNA I (1:500), rat anti-CTIP2 (1:500; Abcam, ab18465), rabbit anti-Ki67 (1:100; EMD Millipore, AB9260), mouse anti-Iba1 (1:200; EMD Millipore, MABN92), rabbit anti-Iba1 (1:500 Wako Chemicals USA Inc.), and rabbit anti-CD31 (Abcam, ab28364). Subsequently, samples were washed with phosphate-buffered saline (PBS) and incubated with secondary antibodies: goat anti-mouse Alexa Fluor 488 (1:500; Abcam, ab150113), goat anti-rat Alexa Fluor 488 (1:500; Abcam, ab150157), and goat anti-mouse Alexa Fluor 546 (1:500; AP192SA6) (Thermo Fisher Scientific, USA). In those sections where IB4 (1:50; Vector Laboratories, FL1201) was used, it was incubated with secondary antibodies for 2 hours. Nuclei were stained with DAPI (0.5 μg/ml) for 20 min. Images were acquired with a TCS SP8 confocal microscope (Leica, Germany) and a laser scanning confocal microscope (Olympus FV1000, Japan) with 20×, 40×, or 63× objective high numerical apertures.

In all cases, at least three brains of each experimental group were examined and analyzed. Brains in each group belong at least to two different litters. For each brain, three consecutive sections were analyzed, and an average of their results was taken as the representative value for the specimen. All data collection was performed by one of the authors (J.B.-A.) using a blinding code. Previous to definite data collection, we analyzed intraobserver repeatability in a subsample of images and confirmed consistency in marker identification. Analyses of the acquired images were carried out using Fiji software (43). We performed a quantitative analysis based on the work by Wu et al. (32) where the band thickness of positive-stained cells was analyzed. Here, we measured the distance between the ventricular limit and the most upper located Ki67+ cells. For CTIP2 staining, we counted the absolute number of cells that were positive in an area with a height of 100 μm and a width of 70 μm.

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