The local immune response to treatment was assessed by characterizing immune cell infiltrates within brain sections collected from high-dose H-FIRE ablation groups at the study endpoint. Tissue samples were sectioned and stained for presence of T cells (CD3, Dako), helper T cells (CD4, Origene), cytotoxic T cells (CD8, Invitrogene), regulatory T cells (FoxP3, Invitrigene), B cells (CD79a, Santa Cruz), microglia (IBA-1, FUJIFILM), M1 macrophages (CD86, Abcam), and M2 macrophages (CD163, Abcam). Methods were followed as presented by Koshkaki et al. (35 (link)). Samples were categorized as peritumoral region (healthy tissue), transition zone (submillimeter zone between the tumor mass and the peritumoral region), tumor mass, and the necrotic core. Tissue samples were imaged using a Nikon Eclipse Ni-U microscope using a Ds-Ri2 camera. The acquired images were analyzed using the NIS element BR software version 5.21.01.
Briefly, auto thresholding was done on all images followed by greyscale conversion. Mean gray values (MGVi) and gray area fractions (AF) were calculated using the software. The values were then used to find the final chromogen intensity (f) as follows:
Mean f and AF values were next calculated using five high power fields from each slide. The immunohistochemistry (IHC) score was calculated using the mean values and the following equation:
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