Avidin biotin peroxidase

The detailed procedure for immunohistochemistry has been described elsewhere (Shih et al., 2016 (link)). In short, coronal sections (30 μm thickness) of the right hemisphere were blocked with goat serum (3% in PBS/0.5% Triton X-100) for 1 h, and probed with the following primary antibodies: pS412-tau (1:1000, AS-55418P, Anaspec), rabbit anti-ionized calcium binding adaptor molecule 1 (Iba1) (1:1000, 091-19741, Wako, Japan) for microglia and biotinylated goat anti-mouse IgG (1:1500, BA-9200, Vector Laboratories, Burlingame, CA, United States) for BBB leakage. The floating sections were incubated with primary antibodies for 16 h at 4°C, then incubated with appropriate secondary antibodies (Vector Laboratories) and avidin-biotin peroxidase (Vector) using 3, 3′-diaminobenzidine as the substrate (Shih et al., 2016 (link)). A parallel section stained without primary antibody served as the negative control.

Slides of bone tissue were allowed to warm to room temperature, then fixed in 10% neutral-buffered formalin. Membranes in the tissue were permeabilized using 0.25% Triton X-100 (catalog no. 9002-93-1; Sigma-Aldrich, Darmstadt, Germany). Endogenous peroxidase activity was quenched using 3% hydrogen peroxide (Solarbio, Beijing, China), and antigens were retrieved using citrate buffer on a hot plate at 85°C. Non-specific reactivity on slides was blocked by incubating them in 5% bovine serum albumin at room temperature, then primary antibody (diluted 1:100) was added for overnight incubation at 4°C. Next, secondary antibody was applied for 60 min at room temperature. Slides were rinsed with 1 × Tris-buffered saline containing 0.5% Tween-20 (TBST), incubated for 30 min with avidin–biotin peroxidase (diluted 1:200), rinsed again in 1× TBST, then developed using 3,3-diaminobenzidine (Vector Laboratories, Burlingame, CA, United States). Slides were examined using an Olympus BX40 light microscope and photographed at ×20 magnification.

For immunohistochemistry (IHC), sections of pancreas (4 µm) were stained with antibodies against insulin (Cell Signaling, Danvers, MA, USA) as described previously [5 (link)]. The slides were incubated in biotin-conjugated secondary antibody (IgG, 1:200 dilution) and avidin-biotin peroxidase (Vector Laboratories, Burlingame, CA, USA) at room temperature for 1 h each. Negative controls were run in parallel without primary antibody in the incubation. The results of the immunohistochemical staining were captured by a microscope with an imaging capture system (Nikon NIS-Elements BR) and analyzed and calculated with ImageJ (Opensource Java Imaging Processing Program by NIH http://imagej.nih.gov/ij/).

For morphological analysis, forebrains were obtained and post-fixed in 4% paraformaldehyde for 72 h at 4 °C after perfusion. Thereafter, samples were cryoprotected with 30% sucrose solution at 4 °C. The 30% sucrose solution was renewed twice in 7 days. These brain samples were then sliced with a freezing microtome at 30 μm. Brain slices were collected in cryoprotectant as previously described [6 (link)] and stored at −20 °C for further immunohistochemical staining. In short, the paraformaldehyde fixed brain sections were washed with PBS, followed by permeabilization for 5 min with 0.1% Triton X 100 in 0.1% sodium citrate. After washing, the sections were stained at room temperature overnight with mouse anti-Ki67 antibody (1:1000, Ab16667, Abcam) for newly proliferated cell or goat anti-doublecortin (DCX, 1:1000, Ab18723, Abcam) for the immature neurons. Then, the specimens were rinsed three times in PBS to wash out non-binding antibodies. Thereafter, the brain specimens were incubated with the appropriate peroxidase-conjugated secondary antibody (Vector, Burlingame, CA, USA) for 2 h at room temperature and then rinsed three times in PBS. The specimens were then incubated with an avidin–biotin peroxidase (Vector) using 3,3′diaminobenzidine as the substrate. After washing three times in PBS, the specimens were imaged using an Olympus light microscope (BX51, Tokyo, Japan).

These were performed as previously reported^{41 (link),42 (link)}. The gastrocnemius muscles from ischaemic and non-ischaemic hind-limbs were collected and embedded in OCT compound (ProSciTech), frozen, and cut into 5 µm-thick sections. The slides were fixed at −20 °C in 95% ethanol for 1 hr. Slides were washed three times in cold PBS with 1% horse serum (5 min/wash) and blocked overnight with 5% horse serum in PBS at 4 °C. Immunohistochemistry was performed using primary antibodies against CD31 (1:100 dilution; Abcam) and smooth muscle α-actin (α-SMA, 1:200 dilution; Abcam). Bound primary antibodies were detected by using appropriate secondary antibodies (biotinylated anti-goat IgG and biotinylated anti-Rat IgG, all at 1:100 dilutions, Vector Labs) using avidin-biotin-peroxidase (Vector Labs) as described previously^{43 (link)}. Pictures from four random areas of each section and three sections per mouse were taken by using a digital camera (Nikon Eclipse Sci epifluorescence microscope, Nikon Corporation) at 40× magnification. Capillary density were quantified by measuring the percentage of CD31 and α-SMA staining out of the total area as previously described.

Dexmedetomidine was obtained from the Jiangsu HengRui Pharmaceutical Co. Ltd. (Jiangsu, China). Rabbit polyclonal anti-c-Fos antibody was purchased from Abcam (Cambridge, MA, United States). Biotinylated donkey anti-rabbit IgG and avidin-biotin-peroxidase were purchased from Vector Laboratories (Burlingame, CA, United States). Finally, 3, 3-diaminobenzidine-tetra-hydrochloride (DAB) was purchased from Sigma-Aldrich (St. Louis, MO, United States). Dexmedetomidine was dissolved in sterile saline before use.