Vectastain elite abc peroxidase kit standard

For immunohistochemistry (IHC), brain sections were immunostained with the following primary antibodies: rabbit polyclonal anti-IBA1 (marker of microglia, Wako Chemicals, Richmond, VA, USA, cat. No. 019–19,741), rabbit polyclonal anti-GFAP (marker of astrocytes, Dako, Glostrup, Denmark, cat. No. Z0334), and rabbit polyclonal anti-albumin (to detect blood brain barrier damage, Abcam, Cambridge, UK, cat. No. ab19196). Sections were then incubated with biotinylated secondary antibody (goat anti-rabbit, Vector Laboratories, USA, cat. No. VC-BA-1000-MM15), labelled by the avidin-biotin-peroxidase procedure (VECTASTAIN® Elite ABC-Peroxidase Kit Standard, Vector Laboratories, USA, cat. No. VC-PK-6100-KI01). The immunoreaction was visualized with 3,3′-diaminobenzidine (DAB, Peroxidase DAB Substrate Kit, Vector Laboratories, USA, cat. No. VC-SK-4100-KI01) substrate and sections were counterstained with Mayer’s haematoxylin (Diapath, Martinengo (BG), Italy, cat. No. C0302). Outcome assessors were blinded to the study groups.

For histopathological examination, samples were fixed in 10% neutral buffered formalin (NBF) for at least 24–48 hours, processed with a Tissue Processor Leica ASP300 S, and paraffin embedded (Embedding Center Leica EG1160). Sections of 4 μm were cut, stained with Haematoxylin-Eosin (H&E) and evaluated under a light microscope (Leica DM 2500). Representative images were captured with a digital camera (Leica DFC310 FX).
For immunohistochemistry 4 μm serial sections from each sample were immunostained with the primary antibodies listed in Supplementary Table 1 and incubated with appropriate biotinylated secondary antibody: goat anti-rabbit (VC-BA-1000-MM15, Vector Laboratories, USA). Sections were labelled by the avidin-biotin-peroxidase (ABC) procedure with a commercial immunoperoxidase kit (VECTASTAIN® Elite ABC-Peroxidase Kit Standard, VC-PK-6100-KI01, Vector Laboratories). The immunoreaction was visualized with 3,3′-diaminobenzidine substrate (Peroxidase DAB Substrate Kit, VC-SK-4100-KI01, Vector Laboratories) and sections were counterstained with Mayer’s haematoxylin. Negative immunohistochemical controls were prepared by replacing the primary antibody with an irrelevant one and known positive control sections were included in each immunolabeling assay.