Avidin biotin

Tumor vessels were observed on 5-μm cryo-sections by targeting mouse CD31 protein. In order to avoid unspecific background signal, sections were first pretreated with 0.3% H₂O₂ and Avidin/Biotin (Vector labs). Then, sections were incubated with a rat anti—mouse CD31 antibody (clone MEC 13.3; BD Biosciences) followed by a goat F(ab′)₂ anti-rat IgG(H+L)-Biotin (3052–08; Southern Biotech). mCD31 was visualized with the Vectastain Elite ABC Kit (Vector Labs) plus DAB chromogenic substrate (Interchim). Sections were counterstained with hematoxylin and mounted with ImmunoMount (Thermo Scientific).

Immunohistochemical analysis was performed as described before [22 (link)]. The material was routinely fixed in 4 % formaldehyde solution and embedded in paraffin. After slicing into 4-μm-thick sections, the preparations were dewaxed in xylene and then rehydrated. Endogenous peroxidase activity was blocked by 3 % hydrogen peroxide in methanol for 30 min. After a short rinse with phosphate buffered saline (PBS), sections were pre-incubated with avidin-biotin (Vector Laboratories, Peterborough, UK; SP-2001) for 15 min to reduce non-specific background staining. The preparations were covered with normal goat serum for 20 min and then incubated with the primary antibodies (CD44, mouse monoclonal, Diagnostic Biosystems, Pleasanton, CA, dilution 1:2000; CD44v6, abcam, Cambridge, MA, dilution 1:1000; and VEGF, mouse monoclonal, Dako, Denmark, dilution 1:50) for 30 min. Then, the sections were washed with PBS, incubated with biotinylated goat anti-mouse immunoglobulin G (BioGenex, Germany) for 30 min and covered with peroxidase-conjugated streptavidin (Dako). The peroxidase reaction was allowed to proceed for 8 min, with 0.05 % 3,3-diaminobenzidine tetrahydrochloride solution as substrate. Slides were counterstained with hematoxylin. Negative controls were also performed by replacing the primary antibodies with mouse or goat ascites fluid (Sigma-Aldrich, St. Louis, MO).

Paraffin sections were deparaffinized with Histoclear and ethanol, antigen was retrieved by incubation in 0.01 M citrate buffer (pH 6.0) at 95 °C for 20 min. Slides were incubated in 0.9% H₂O₂ for 30 min and afterwards placed in blocking buffer (Rabbit IgG, no. PK-6101; Vector Lab) for 30–60 min at room temperature. Livers were further blocked with Avidin/Biotin (Vector Lab, no. SP-2001) for 15 min each. MAFs were washed briefly with PBS and fixed for 10 min with 2% paraformaldehyde dissolved in PBS. Cells were permeabilized for 45 min with PBG. Primary antibodies were applied overnight at 4 °C. Slides were washed three times with PBS and incubated for 30 min with secondary antibody (no. PK-6101; Vector Lab). Antibodies were detected using rabbit peroxidase ABC Kit (no. PK-6101; Vector Lab) according to the manufacturer’s instructions. Substrate was developed using NovaRed (no. SK-4800; Vector Lab) or DAB (no. SK4100, Vector Lab). Sections were counterstained with haematoxylin. For IF, sections were treated as before and after the secondary antibody incubation Fluorescein Avidin DCS (1:500 in PBS, no. A-2011, Vector Lab) was applied for 20 min. For IF on MAFs, Alexa Fluor secondary antibody (1:2,000; Molecular Probes) was applied for 30 min at room temperature. Sections or cells were stained with DAPI for 5–10 min and mounted in vectashield mounting media.

Paraffin embedded tissues were sectioned 4 μm thick. Endogenous peroxidase activity was quenched for 30 min in darkness by 1% H₂O₂ (Sigma Aldrich, St. Louis, MO, USA) in methanol (Histolab, Gothenburg, Sweden). After blocking with avidin-biotin (Vector Laboratories, Burlingame, CA) and normal goat serum (5%) for 60 min, the sections were incubated with primary antibodies in Tris-buffered saline (TBS)/saponin buffer overnight. Biotinylated goat anti-rabbit IgG secondary antibodies were added in 2% normal blocking serum. Slides were developed for 5 min in diaminobenzidine (DAB, VECTASTAIN, Vector Laboratories) and counterstained with Mayer's hematoxylin (Histolab). Images were analyzed using the ScanScope CS GL SS5082 and Aperio ImageScope v11.1.2.760. Positively stained cells were quantified as average number of cells in 10 microscopic fields (20x magnification) by two researchers blinded to the samples.

Paraffin-embedded tissue sections on coated slides were dewaxed in xylene and rehydrated. Frozen sections of mouse tissues were placed directly on slides and then fixed with 10% buffered formalin. All subsequent washes were done with PBS–0.05% Tween 20 (PBS-T). Sections were first blocked in CarboFree (10% normal goat serum for SGRP probing) for 1 h at 37°C. Esterase treatment control slides were exposed to active esterase HE-Fc forms (20 μg/μl) during serum blocking. Sections were further blocked with avidin/biotin (Vector Laboratories, Burlingame, CA) and quenched of active peroxidase activity by the use of Bloxall (Vector Laboratories). Sections were probed with HE-Fc SGRPs (at 20 μg/ml) in complex with biotinylated goat anti-human Fc γ-specific antibody (10:1 molar ratio) overnight at 4°C. Sections were probed with biotinylated plant lectins SNA (Vector Laboratories) and MAH (MAA-II, Vector Laboratories) at 10 μg/ml and 20 μg/ml, respectively, for 3 h at RT. Sections were then exposed to ABC Vectastain strepavidin-HRP, followed by NovaRed HRP substrate (Vector Laboratories). Sections were counterstained in hematoxylin, dehydrated, and embedded in Cytoseal XYL.