Representative rat brain microphotographs and corresponding binary masks supporting significant findings from immunohistochemical analysis in “Morphological Changes in the Rat Brain in the Early Post-Resuscitation Period”
Description
This dataset contains additional representative microphotographs of immunohistochemically stained rat brain sections and the corresponding binary masks used for quantitative histological image analysis. The dataset includes images from three rats in each comparison group for which statistically significant differences in the number and/or intensity of immunoreactive objects were found. The binary masks illustrate the image-processing and quantification procedure used to identify and quantify immunoreactive objects. The staining, image acquisition, and quantitative analysis procedures are described in detail in the associated manuscript "Morphological changes in the rat brain in the early post-resuscitation period: the relationship between hypoxia-sensitive immunohistochemical markers, hemodynamic and acid-base status".
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Paraffin brain sections were processed for immunohistochemical staining using antibodies against PHD1, Iba-1, and S-100β, followed by DAB visualization and hematoxylin counterstaining. Whole-slide digital images of IHC-stained sections were acquired using an Aperio ScanScope CS slide scanner at 20× objective magnification under standardized acquisition conditions. From the digitized brain sections, non-overlapping artifact-free regions of interest were selected in the sensorimotor cortex and hippocampal CA1 and CA4 regions. Quantitative image analysis was performed using NIS-Elements BR software, version 5.20. Positively stained regions were identified by color thresholding in the HSI color space, with threshold values determined separately for each immunohistochemical marker and then applied uniformly to all images stained for the same marker. Binary masks were generated from the corresponding micrographs to identify DAB-positive immunoreactive objects. Artifacts and non-specific staining outside the regions of interest, including blood vessels, were manually corrected. For each image, the number of stained objects and their mean staining intensity were quantified. DAB reciprocal staining intensity was calculated as RSI = 255 − mean intensity.