Immpact novared peroxidase substrate kit

Whole feet were examined by IHC as described previously (11 (link)). Sections were stained with rat anti-mouse Ly6G (catalog number NMP-R14; Abcam, Cambridge, MA, USA) (neutrophils), F4/80 (Abcam) (macrophages), or CD3 (Abcam) (T lymphocytes). Detection used NovaRED secondary antibody (ImmPACT NovaRED peroxidase substrate kit [catalog number SK-4805]; Vector Laboratories, Burlingame, CA, USA). Slides were digitally scanned using an Aperio AT Turbo system (Leica Biosystems), images were analyzed using Aperio ImageScope software (Leica), and cell quantitation was undertaken using open-source image analysis software, QuPath v0.2.3 (25 (link)). Three sections per foot were analyzed to produce a mean value for each mouse.

Tumor tissue with overlying skin was harvested on day 28. Anti-CD4, anti-CD8, Anti-CD49b and anti-F4/80 antibodies (eBioscience) were incubated with tissue sections at 4 °C overnight. Sections were then treated sequentially with secondary antibody (ZytoChem Plus HRP One-Step Polymer anti-mouse; Zytomed, Berlin, Germany) and substrate solution (ImmPACT NovaRED Peroxidase Substrate Kit; Vector, Burlingame, CA, USA). Pulmonary edema was assessed by hematoxylin and eosin staining.

After antigen retrieval, the sections were washed in 1 × PBS (phosphate buffered saline, pH 7) and circled using a DAKO pen (ImmEdge, Vector Laboratories inc.). A droplet of a 1:400 dilution of goat anti-type I collagen antibody (IgG, unlabeled, Southern Biotech) was added to the sections and incubated for 1 h at room temperature. Next, endogenous peroxidases were inhibited by incubating the sections in 0.1% H₂O₂ in methanol for 20 min. The sections were washed in 1 × PBS, then incubated for 30 min with a 1:100 dilution of secondary antibody (rabbit-anti-goat IgG (H + L), Mouse/Rat/Human ads-horse radish peroxidase (HRP) (Southern Biotech) in 1 × PBS with 5% normal mouse serum (NMS), and washed again. After a 30-min incubation with a 1:100 dilution of tertiary antibody [goat-anti-rabbit IgG, IgG(H + L)-HRP, Southern Biotech] in 1 × PBS with 5% NMS, the sections were washed and incubated for 5 min with a Novared reaction mixture (ImmPACT, NovaRED peroxidase substrate kit for laboratory use, Vector Laboratories). The sections were incubated in hematoxylin for 1 min, flushed with tap water for 5 min, and dehydrated in three washes with 100% ethanol. Finally, the dehydrated sections were mounted using DePex (SERVA Electrophoresis GmbH). Pictures of the stained sections were taken using an Olympus BX41 Microscope and Hamamatsu NanoZoomer digital slide scanner.

Tissues were fixed in 4% formaldehyde and embedded in paraffin. Immunostaining was performed using 4 µm thick tissue sections. For iron detection, slides were stained with Perls’ Prussian blue (together with Nuclear Fast Red as counterstain) using standard procedures. For αSMA immune-staining, endogenous peroxidases were neutralized in 3% H₂O₂ for 20 min. Tissue sections were permeabilised for 20 min in PBS containing 0.5% Triton X, then blocked for 30 min at room temperature in PBS containing 3% BSA, 0.1% Triton X-100, and 10% normal goat serum. Sections were incubated at 4 °C over night with primary antibody against αSMA (A5228 from Merck, diluted 1:200 in PBS + 3% BSA, 0.1% Triton X-100, 1% normal goat serum). Immune complexes were detected using a HRP-conjugated secondary antibodies and the ImmPACT NovaRED Peroxidase Substrate Kit (Vector Laboratories, Burlingame, CA, USA) according to the manufacturer’s instructions. Counterstaining was performed with Nuclear Fast Red (Vector Laboratories). Images were acquired using the Lamina Slide scanner (Akoya Perkin Elmer) and analysed with Case Viewer software (3DHistech).