Biotinylated goat anti rat igg

The avidin–biotin immunoperoxidase method was used for deparaffinized zinc formalin-fixed, paraffin-embedded sections. Specific methods are detailed in our previous article (27 (link)). The primary antibodies including CD11b (1:5,000, ab133357, Abcam), F4/80 (1:200, 123101, BioLegend), CD68 (1:500, GB11067, Servicebio), Cleaved caspase-3 (1:300, 9661, Cell Signaling), BrdU (1:50, B44, BD), Ki67 (1:400, 12202, Cell Signaling), CD8 (1:500, GB11068, Servicebio), granzyme-B (1:200, sc-8002, Santa Cruz), followed by incubation with secondary antibodies: biotinylated goat anti-mouse IgG (1:300, BA-9200, Vector), biotinylated goat anti-rat IgG (1:300, BA-9400, Vector) and biotinylated goat anti-rabbit IgG (1:300, BA-1000, Vector). Images were taken through a light microscope (Olympus).

DAB immunostaining: Brains from 4 % paraformaldehyde perfusion-fixed SEZ6 TKO (n = 4) and WT (n = 7) adult mice were cryosectioned and underwent sequential incubation in BLOXALL (Vector Laboratories), 4 % Bovine Serum Albumin (BSA, Sigma Aldrich) and 0.1 % Triton X-100 (Sigma Aldrich) in phosphate buffered saline (PBS), and avidin/biotin (Avidin/Biotin Blocking Kit, Vector Laboratories). Sections were incubated overnight with monoclonal rat anti-SEZ6 or SEZ6L primary antibodies diluted in 2 % BSA and 0.3 % Triton X-100 in PBS. Sections were washed with PBS, incubated with biotinylated goat anti-rat IgG (Vector Laboratories) and processed using the VECTASTAIN ABC Kit (Vector Laboratories) and ImmPACT DAB peroxidase substrate as chromogen (Vector Laboratories) according to manufacturer’s instructions. Some sections were counterstained with haematoxylin. Primary or secondary antibodies were omitted on sections in each experiment to confirm staining specificity. Low power images were acquired on a Mirax slide scanner and high power images were acquired at 63× magnification on a Zeiss Axio microscope.

The procedures for immunohistochemistry were carefully performed under the same conditions to evaluate differences between the groups as described in previous studies [9 (link),25 (link)]. The brain sections were rehydrated in graded concentrations of alcohol and quenched endogenous peroxidase activity with 0.3% hydrogen peroxide (H₂O₂) in phos-phate-buffered saline (PBS). For retrieving antigens in tissues, the brain sections were heated in citrate buffer (pH 6.0) with the retriever (2100 Antigen Retriever, Prestige medical, Lancashire, UK). Additionally, the slides were then cooled at 25 °C and washed in PBS. For protein blocking, the sections were incubated in 5% normal goat serum for 30 min. For primary antibody binding, the tissues were incubated in diluted rat anti-SST (1:100, EMD Millipore, Temecula, CA, USA), rabbit anti-Ki67 (1∶500; Abcam, Cambridge, UK, a marker for proliferating cells), or rabbit anti-doublecortin (DCX, Abcam, a marker for developing neuroblasts) antibodies for 48 h at 4 °C. Thereafter, they were exposed to biotinylated goat anti-rat IgG or goat anti-rabbit IgG (1:200, Vector Laboratories, Burlingame, CA, USA), streptavidin peroxidase complex (Vector Laboratories, Burlingame, CA, USA), and then visualized with 3,3′-diaminobenzidine tetrahydrochloride (Sigma-Aldrich) in 0.1 M Tris-HCl buffer (pH 7.4).

Mice were perfused with EM grade 4% PFA. One hemisphere of the brain was sectioned at 50 μm, coronally on a vibratome (Leica VT1000S, Leica Inc., Nussloch, Germany), and post-fixed in 2.5% glutaraldehyde for 2 h at RT. Immunocytochemistry was performed using anti-Ctip2 antibody (1:500, Thermo Fisher Scientific, Rockford, IL, USA), and biotinylated goat anti-rat IgG (1:500, Vector Laboratories, Burlingame, CA, USA). Samples were processed for prepared for EM as previously reported [14 (link)].

Liver tissues were collected and stained with hematoxylin and eosin, and light microscopy was used to observe inflammation and tissue damage. Liver macrophages and neutrophils were detected using primary rat anti-mouse F4/80, CD11b, and Ly6G mAb, respectively (BD Biosciences, San Jose, CA, USA). The secondary, biotinylated goat anti-rat IgG (Vector, Burlingame, CA, USA) was incubated with immunoperoxidase (ABC Kit, Vector), according to the manufacturer’s instruction. Positive cells were counted blindly in 10 HPF/section. iNOS and CD206 in KCs were identified by immunofluorescence using rabbit anti-mouse iNOS mAb and anti-mouse CD206 mAb (Cell Signaling Technology, MA, USA). After incubation with secondary goat anti-mouse Texas Red-conjugated IgG (Sigma, St.Louis, MO, USA), KCs were premounted with VECTASHIELD medium with DAPI (Vector). Negative control slided with the primary antibodies omitted were included in all assays. Positive cells were blindly observed in 10 HPF/section (×200).