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. 2019 Apr;369(1):9–25. doi: 10.1124/jpet.118.254854

Fig. 11.

Fig. 11.

Immunohistochemical analysis of the effect of PNA5 on Iba-1 levels in the brain. Quantitative analysis of Iba-1 immunohistochemical staining in the brain meningeal surface in HF-PNA5, HF-saline, and control animals 10 days after the last injection of PNA5 or saline. Daily treatment with PNA5 for 3 weeks inhibited Iba-1 immunohistochemical expression found within the meningeal surface layer. Importantly, this effect persisted 1 week after the end of PNA5 treatment, even while HF was still present. The level of DAB staining was quantified with NIH ImageJ software. The ROI manager in ImageJ was used to create a standard-sized ROI, and this same size ROI was used to analyze each slice. Identical image acquisition settings were used for all images. To ensure a uniform background across sections, we used ImageJ background subtraction prior to thresholding the images. We used an identical standard ImageJ thresholding tool setting for each image acquired. These image acquisition control techniques allowed us to successfully quantify staining that was duplicated in every image that was compared. From the threshold image, integrated density measurements from each ROI were obtained. Differences between groups were analyzed using a Student’s t test (*P < 0.05).