Rat anti cd11b αm integrin subunit

Five sections from each brain (400 μm apart) were rehydrated in distilled water and stained using immunohistochemistry as previously described (7 (link), 27 (link), 29 (link), 30 (link)). Briefly, the sections were incubated overnight with rat anti-CD11b αM integrin subunit (1:5,000, Serotec, UK) or rabbit polyclonal anti-glial fibrillary acidic protein (GFAP) (1:6,000, DAKO, UK), primary antibodies, for 1 h with biotinylated goat anti-rat or -rabbit (1:100, Vector, UK) secondary antibodies, followed by incubation with Avidin-Biotinylated horseradish peroxidase Complex (Vector, UK) and visualization with diaminobenzidine/H₂O₂ (Fisher Scientific, UK) (7 (link), 27 (link), 29 (link), 30 (link)).
Five further sections from each brain with the same spacing were stained using Terminal transferase mediated d-UTP nick end labeling (TUNEL) (Roche, UK). The staining procedure followed the manufacturer protocol with Co/Ni enhancement (7 (link), 27 (link), 29 (link), 30 (link)).
Five more sections per brain with the same spacing were stained with Cresyl-Violet (Nissl).

Five sections from each brain (400um apart), were rehydrated in distilled water and stained using immunohistochemistry as previously described [20 (link)]. Briefly, the sections were incubated overnight with rat anti-CD11b αM integrin subunit (1:5000, Serotec, UK) or rabbit polyclonal anti-GFAP (1:6000, DAKO, UK) primary antibodies, for 1h with biotinylated goat anti-rabbit or -rat (1:100, Vector, UK) secondary antibody, followed by incubation with Avidin-Biotinylated horseradish peroxidase Complex (Vector, UK) and visualisation with diaminobenzidine/H₂O₂ (Fisher Scientific, UK).
Five further sections from each brain with the same spacing were stained using Terminal transferase mediated d-UTP nick end labelling (TUNEL) (Roche, UK). The staining procedure followed the manufacturer protocol with Co/Ni enhancement.
Five more sections per brain with the same spacing were stained with cresyl violet (Nissl).
All sections belonging to the same hypothermia setting were stained simultaneously to avoid differences in intensity.