Ihc p

Immunohistochemical staining with rabbit polyclonal anti-Bax (ab53154), anti-Bcl2 (ab59348), and anti-caspase-3 (ab44976) primary antibodies (IHC-P, at 1:100 dilution, Abcam^®, Cambridge, MA, United States) was performed via the Dako automated system (EnVision FLEX Peroxidase-blocked), as previously described (El Asar et al., 2019 (link)). In addition, the goat anti-rabbit IgG H&L (HRP) was added as the secondary antibody. Negative controls were obtained by omitting the primary antibodies in the automated staining protocol. The area percentage of the positive immune reaction of Bax, Bcl2, and caspase-3 was estimated using Leica Qwin 500C, Leica Image Analysis System Ltd. (Cambridge, England), in 10 live random fields (×400 magnification) from each group. The sections were evaluated using a Leica ICS150 HD microscope camera (Leica Imaging, Cambridge, United Kingdom).

Immunohistochemical assessment of the macrophage phenotype markers CD8, CD68, and CD 163 and the microglial marker Iba-1 was performed using the avidin–biotin peroxidase method as described previously (Magdy et al., 2021 (link)). Briefly, paraffin-embedded brain sections were dewaxed in xylene, rehydrated in a decreasing alcohol gradient, and microwave-treated (0.01 M trisodium citrate), for antigen retrieval. Quenching of the endogenous peroxidase activity was achieved via incubation in 10% Hydrogen peroxide. The sections where then incubated with the primary antibodies, as follows: anti-CD8 antibody (Thermo Fisher Scientific, MA5-13473, 1:25), rabbit polyclonal anti-ionized calcium-binding adapter molecule 1 (IBA1) antibody (ab153696, 1:500, Abcam®), rabbit monoclonal anti-CD86 antibody [C86/2160R] (ab234401, 1:50, IHC-P, Abcam®), and anti-CD163 antibody [EPR19518] (ab182422) (1:100, Abcam®, Cambridge, MA, United States). Staining was completed by incubation with the substrate-chromogen 3,3′- diaminobenzidine (DAB) and counterstaining with hematoxylin. The mouse spleen tissue was used as the positive control, whereas the negative controls were obtained through omission of the primary antibodies in the automated staining protocol.