Tcs spe dmi 4000b

One uterine horn per mouse was fixed overnight in 10% neutral buffered formalin, processed in increasing concentrations of ethanol, and paraffin embedded. Tissues sections (5μm) were processed for histological analysis. Sections were stained with Hematoxylin and Eosin and analyzed by two pathologists blinded to the experimental exposures. For immunohistochemistry, sections were incubated with antibody specific to phospho-histone H3 (pHH3), alpha smooth muscle actin (αSMA) and cytokeratin 8 (CK8) (S1 Table), then with goat HRP-conjugated secondary antibody (Santa Cruz Biotechnology, 1:1000). Slides were developed for 5 minutes with a Vector DAB Kit (Vector Laboratories) per manufacturer's protocol, counterstained in CAT Hematoxylin. Representative images were taken on a Nikon Eclipse E800 upright microscope with an Olympus DP71 camera and manufacturer's software. We used primary antibody against pHH3 for immunofluorescence (S1 Table) followed by goat Alexa Fluor 488—conjugated secondary antibody (Molecular Probes, 1:1000). Sections were counterstained with DAPI imaged by confocal microscopy (Leica TCS SPE/DMI4000B; Leica Application Suite software).

Cultured cortical neurons were imaged on a fluorescence microscope (DM6000 B, Leica) with a ×40-oil objective using Leica software for quantifications. Representative images of cultured cortical neurons were taken using an SP5 confocal microscope with a ×40-oil objective and a 2.5 digital zoom factor using Leica software. Fluorescent tissue slides stained for NeuN were scanned on a Zeiss AxioScan Z1 (Histopathology core facility in Cancer Research UK—Cambridge institute) using a ×20 light objective. Images containing the regions of interest were exported in TIFF format. All images within an experiment were taken with identical settings when the eGFP intensity was quantified. Representative images for astrocytes, microglia, the oligodendrocytes lineage, neurons with PNNs and the dCST were taken using laser-scanning confocal microscopy (Leica, TCS SPE, DMI4000B).

Slides were analyzed using a confocal Leica TCS SPE DMI 4000B (LAS X software, Leica, Wetzlar, Germany) microscope. Z-stack images were acquired in 0.33 µm sections and processed using the 3-D volume setting of LasX software. Images were processed using Adobe Photoshop CC 2018 and FIJI-ImageJ-64 software. Heat maps were generated from red and green channels using ImageJ software (Analyze> Colocalization> colocalization threshold > show colocalized pixel map), then channels were split and co-localization channel assigned LUT > Fire and auto-contrasted. Quantitation of p-PKCζ–T560 and F-actin were accomplished with ImageJ. Green and red channels were analyzed separately. A 35-pixel length line was drawn and centered over cell–cell contacts or pedestals, and the line duplicated in each channel (Edit > selection > restore selection). Pixel intensity was measured over the length of the line (Analyze > plot profile), recorded for at least 15 regions of interest from membranes and pedestals of three biological replicates and statistical analysis was performed as described below.