Biotinylated goat anti mouse igg

Gal-1 staining was performed in 5 μm sections of paraffin-embedded lung tissue. Briefly, after being rehydrated in an alcohol gradient, the floating sections were rinsed and treated with 0.3% hydrogen peroxide and 0.5% Triton X-100 in PBS for 0.5 hours to quench endogenous peroxidase activity. Then, following a blocking step with 5% normal donkey serum (Vector Laboratories, Burlingame, CA) in PBST (PBS containing 0.5% Triton X-100) for one hour at 37°C, the sections were incubated with primary antibodies of mouse anti-galectin-1 (Santa Cruz Biotechnology, CA, USA) diluted in 1 : 100 in PBST overnight at 4°C. Amplification was performed with biotinylated goat anti-mouse IgG (1 : 200; Vector Laboratories) in PBST for 1 hour at 37°C. Positive staining was detected using a peroxidase-conjugated streptavidin complex, and color was developed using 0.05% 3,3′-diaminobenzidine (Sigma-Aldrich). The sections were mounted on glass slides in TB containing 0.5% gelatin and air-dried overnight before being dehydrated in ethanol-xylene, and coverslips were added using Permount. Photographs were taken using a Zeiss Axioskop 2 microscope (Carl Zeiss).

Monoclonal anti-CD63 antibody (clone TS63) was from Abcam (Cambridge, UK) and biotinylated goat anti-galectin-3 (gal-3) antibodies from R&D Systems (Minneapolis, MN, USA); 3,3′,5,5′-tetramethylbenzidine (TMB), bovine serum albumin (BSA), D-lactose, and methyl- alpha D-mannopyranoside were from Sigma (St. Louis, MO, USA). Biotinylated goat anti-mouse IgG, biotinylated plant lectins, concanavalin A (ConA), SNA (Sambucus nigra agglutinin), and the Elite Vectastain ABC kit were from Vector Laboratories (Burlingame, CA, USA). Sephadex G 200 and Sephadex DEAE A-50 were from Pharmacia AB, Uppsala, Sweden. The silver stain kit and SDS-PAGE molecular mass standards (broad range) were from Bio-Rad (Hercules, CA, USA). Nitrocellulose membrane and Pierce ECL Western Blotting Substrate were from Thermo Scientific (Rockford, IL, USA). BCA Protein Quantification Kit was from Abcam (Cambridge, UK). Microwell plates were from Thermo Scientific (Roskilde, Denmark). Lymphocyte separation medium LSM-B was from Capricorn Scientific GmbH (Ebsdorfergrund, Germany).

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).

Paraffin sections were routinely deparaffinized in xylene and rehydrated in decreased alcohol gradient. After a brief wash with deionized water, tissue antigen was retrieved through heating in sodium citrate buffer for 15 min, followed by incubation with 3% H₂O₂ for 10 min to quench the endogenous peroxidase. Following three times of PBS washing, non-specific antigen-binding sites were blocked with 10% goat serum in PBS (Gibco) overnight. The sections were then incubated with anti-Sox9 (1:5000, EMD Millipore, Cat# AB5535); anti-ki67(1:500, BD, Cat# 550609); anti-MMP-7 (1:100, Cell Signaling Technology, Cat# 3801S); anti-Olfm4 (1:500, Cell Signaling Technology, Cat# 39141S) at 4 °C overnight. After washing in PBS, the sections were incubated with biotinylated goat anti-rabbit IgG (1:1000, Vector Cat# BA-1000) or biotinylated goat anti-mouse IgG (1:1000, Vector Cat# BA-9200) for 2 hr. in room temperature. The detection was performed with a DAB detection kit (Vector Laboratories, Cat# SK-4100) and a VECTASTAIN kit (Vector Laboratories, Cat# PK-7100) according to the manufacturer’s instructions. Following counter-staining in Hematoxylin (Solarbio® LIFE SCIENCE Cat# H8070) and mounting with neutral balsam (Solarbio® LIFE SCIENCE Cat# 96949–21-2). Sections were observed using a light-field microscope (Leica DMI3000B).

Free-floating sections were pretreated with 1.66% H₂O₂ for 30 min to quench the endogenous peroxidase activity and then for 1 h in PB with 0.25% Triton-X and 3% normal goat serum (Vector Laboratories). The sections were then incubated overnight at 4 °C with a mouse antiPHF-tauAT8 antibody (Pierce Endogen, 1:2000), and the following day they were rinsed and incubated for 1 h in biotinylated goat anti-mouse IgG (1:200; Vector Laboratories). Antibody binding was detected with a Vectastain ABC immunoperoxidase kit (Vector Laboratories) and visualized with the chromogen 3,3′-diaminobenzidine tetrahydrochloride (DAB; Sigma-Aldrich). After staining, the sections were dehydrated, cleared with xylene, and covered-slipped (DePeX; Merck KGaA 100579).

We used the following primary antibodies from NeuroMab (UC Davis/NIH, Davis, CA, USA): (1) monoclonal anti-Kv4.2 (K57/1; amino acid. 209–225 of human Kv4.2, Q9NZV8), (2) monoclonal anti-Kv4.3 (K75/41; aa. 415–636 of rat Kv4.3, Q62897), (3) monoclonal anti-KChIP3 (K665/38; amino acid 1–256 of rat KChIP3, Q9JM47) and (4) monoclonal anti-KChIP4 (K55/7; amino acid 1–233 of rat KChIP3, Q3YAB7). The characteristics and specificity of these antibodies using the corresponding knockout mice have been described by the manufacturer (http://neuromab.ucdavis.edu/catalog.cfm, UC Davis/NIH, Davis, CA, USA). Other primary antibodies used were the following: (1) rabbit anti-Kv4.2 polyclonal (APC-023; Alomone Labs., Jerusalem, Israel), (2) rabbit anti-Kv4.3 polyclonal (APC-017; Alomone Labs., Israel) and (3) rabbit anti-KChIP3 polyclonal (Rb-Af400; aa. 239–256 of rat KChIP3, MM_032462; Frontier Institute Co., Sapporo, Japan) antibodies.
We used the following secondary antibodies: (1) biotinylated goat anti-mouse IgG and biotinylated goat anti- guinea pig IgG (Vector Laboratories, Burlingame, CA, USA) and (2) goat anti-guinea pig and anti-mouse IgG coupled to 1.4 nm gold (1:100; Nanoprobes Inc., Stony Brook, NY, USA).

Some series of chicken embryonic brain sections (E8–E12) were processed for immunohistochemistry, following a procedure previously described (Abellán and Medina, 2009 (link)).
In order to detect radial glial fibers in chicken, we used a monoclonal antibody against chicken vimentin (H5 from Developmental Hybridoma Bank, Iowa, USA; Herman et al., 1993 (link)). The specificity of this antibody has beed shown by the manufacturer using Western blot (labeling a band of roughly 52 kDa, corresponding to the protein vimentin).
The immunohistochemical procedure was as follows. After washing in PBS, the sections were incubated in the primary antibody, diluted 1:50 in PBS containing 0.3% Triton X-100, for 2 days at 4°C, under constant and gentle agitation. Then, the sections were washed and incubated in a secondary antiserum for 1 h at room temperature (biotinylated goat anti-mouse IgG; diluted 1:200; Vector, Burlingame, CA, USA). Following this, the sections were washed and incubated in the avidin-biotin complex (ABC kit; Vector; 0.003% dilution) for 1 h at room temperature. Finally, the immunolabeling was revealed by 0.05% diaminobenzidine (DAB; Sigma-Aldrich, Steinheim, Germany) in 0.05 M Tris buffer (pH 7.6), containing 0.03% H₂O₂.