Anti rat ig hrp detection kit

Two central brain slices with the highest radiotracer concentration from autoradiography, as given by autoradiography Conc_mean, per subject were selected for histological analysis. Paraffin embedding, slicing into 5 μm sections and staining for hematoxylin and eosin (H&E) procedures were performed by UAB histology core facility 71 (link). Slices were dewaxed using EZ-DEWAX (HK584, BioGenex, Fremont, CA) twice for 5 minutes, followed by antigen retrieval with citrate buffer using an EZ-Retriever System (MW015-IR, BioGenex, Fremont, CA) and blocking with 5% BSA in TBST for 5 minutes at room temperature. CD8a monoclonal primary antibody (1:100, 4SM15, Invitrogen, Waltham, MA) was incubated overnight at 4 ºC. Anti-Rat-Ig HRP Detection kit (51-7605KC, BD Pharmingen, Franklin Lakes, NJ) was used for the secondary antibody (1:100). DAB substrate (SK-4100, Vector Laboratories, Newark, CA) was incubated for 7 minutes at room temperature for the development of the stain. Scanning of H&E and IHC stained slides was performed using an EVOS M7000 Imaging System (Thermo Fisher, Waltham, MA) at 20x magnification.

For immunofluorescent, the cryosection (10 μm) was fixed with 4% paraformaldehyde, and pemeabilized with 0.1% Triton X-100. After blocking with 1% bovine serum albumin, antibodies to PDPN (clone: D2-40, DAKO, Glostrup, Denmark) and Fibrinogen (1:100) (Abcam) were incubated overnight. The slides were subsequently stained with secondary antibodies; 4 μg/ml of Alexa Fluor 594 anti-mouse IgG (H + L) and Alexa Fluor 488 anti-rabbit IgG (H + L) (Thermo Fisher Scientific), respectively. The mounted slides with ProLong Diamond Antifade Mountant with DAPI (Thermo Fisher Scientific) were examined using BioRevo BZ-9000 (Keyence, Osaka, Japan). For immunohistochemistry, the cryosection (10 μm) was fixed in ice-cold acetone and endogenous peroxidase was blocked with 0.3% hydrogen peroxide in methanol. After blocking with 10% goat serum, an antibody to CD41 (1:100) (clone: MWReg30, GeneTex, Irvine, CA, USA) was incubated overnight. Colouring was performed with Anti-Rat Ig HRP Detection Kit (BD Pharmingen). Mayer’s hematoxylin solution (Wako) was used for counterstain. All images were taken with BioRevo BZ-9000 (Keyence). CD41-positive area was analysed by BZ-II Analyzer (Keyence).

Mounted tissues were deparaffinized in an EZ-DEWAX bath two times for 5 min. Antigen retrieval was achieved by heating in citrate buffer (Thermo Fisher) for 10 min at 100 °C. Slides were then cooled at room temperature and blocked with 5% BSA (bovine serum albumin) in TBST (Tris buffered saline containing Tween 20) for 5 min at room temperature. Slides were incubated with anti-mouse CD8a monoclonal antibody (clone 4SM15, eBiosciences, catalog #14-0808-82) at 5 µg/mL, followed by anti-rat IgG Biotin, Streptavidin HRP (anti-Rat Ig HRP Detection Kit, BD Biosciences, catalog #551013), and DAB visualization. Nuclei were counterstained with hematoxylin. Control slides were stained as above, except the primary anti-mouse CD8 antibody was omitted. Stained slides of interest were digitalized using a Path Finder Enabler IV Slide Scanner (Electron Microscopy Sciences).