Diaminobenzidine dab

20 μm thick brain sections were cut on a cryostat. Brain sections (free float) were incubated overnight with the following antibodies: rpS6 (1:500) and phospho (Ser 240 and 244) rpS6 (1:500). The sections were subsequently incubated with a biotinylated anti-mouse IgG (Vector Laboratories, Burlingame, CA,) at 1:200 for 1 hr and then incubated with an avidin-biotin peroxidase complex solution (Vector Laboratories, Burlingame, CA) at 1:100 for 1hr. After each incubation, the sections were rinsed for 15 min with PBS containing 0.3% Triton X-100. Finally, the products of the immunoreaction were visualized using diaminobenzidine (DAB) as the substrate (Vector Laboratories, Burlingame, CA). To evaluate the ischemic core, the transition area and peri-infarct area, sections stained with cresyl violet were compared to those that were immunostained.

The 6-month-old mice were perfused with 0.1 M phosphate buffer (PB) followed by fixative solution (2% paraformaldehyde, 2.5% glutaraldehyde in 0.1 M PB pH 7.4). The brains were extracted and stored in the same fixative solution for 3 days at 4°C. Then the brains were cut into 70 μm slices by vibratome. The sections were then subjected to Diaminobenzidine (DAB) immunostaining following the manual (Vector, USA) using antibody against a human-specific marker STEM121.The selected vibratome sections that contained DAB-labeled cells were post-fixed and processed for electron microscopy (EM) (Chen et al., 2016 ; Jiang et al., 2016 ; Jiang et al., 2013a (link)). Brain sections were washed overnight in PBS, post-fixed for 2 hr at RT with 1.5% K₄Fe(CN)₆ and 1% OsO₄ in 0.1 M PB, and then rinsed and stained with 3% uranyl acetate for 1 hr at 4°C. The sections were further transferred to 35%, 50%, 70%, 90% and 2 steps of 100% ethanol, and 2 steps of propylene oxide for dehydration. Next, the sections were embedded and sectioned on an ultramicrotome (Leica Ultracut UCT, Leica Microsystems). The collected sections were transferred onto grids and stained with uranyl acetate for 15–30 minutes and lead citrate for 3–15 minutes. EM images were captured using a high-resolution charge-coupled device (CCD) camera (FEI).

Free-floating sections were immunostained by overnight incubation in primary antibody (rabbit anti-FOXP1, Abcam, ab16645, 1:50,000 dilution; mouse anti-TH, Millipore, MAB-377, 1:50,000 dilution; rabbit anti-Cre, Novagen, 69050–3, 1:5,000 dilution; rabbit anti-cFos, Millipore, ABE457, 1:5,000 dilution; goat anti-CTb, List Biological Laboratories, 1:50,000 dilution), in PBS with 0.25% Triton-X and 0.05% sodium azide. Tissue was then washed three times in PBS (30s each) and incubated in biotinylated secondary (1:1000 dilution, Jackson Immunoresearch, West Grove, PA) for 30 min, followed by three 30s rinses in PBS, and 1-hour incubation in avidin-biotin complex (Vector Laboratories, Burlingame, CA). Tissue was then rinsed in sodium acetate buffer (0.1M, pH 7.4), followed by incubation for 5 min in 0.05% diaminobenzidine (DAB, Vector Laboratories) with 0.1% nickel and 0.01% hydrogen peroxide, revealing a blue-black reaction product. For florescent staining, Cy3-conjugated anti-rabbit and 488-conjugated anti-goat (1:1000 dilution, Jackson Immunoresearch, West Grove, PA) were used. Counterstaining was achieved using deep-red Nissl staining (Fisher, N21483).