Streptavidin horseradish peroxidase

Paraffin-embedded brain tissue of infected and healthy control mice was deparaffinized and boiled in 10 mM citrate buffer (pH 6) in a microwave for antigen retrieval. Endogenous peroxidases were blocked by incubation with 0.3% hydrogen peroxide in methanol (MERCK, Hohenbrunn, Germany). Sections were incubated with anti-CCL20 antibody (Abcam, Cambridge, UK, ab9829) in blocking solution or (vi) with blocking solution without primary antibody (negative control). Bound primary antibody was detected using biotinylated goat anti-rabbit IgG (Vector Labs, Burlingame, CA, USA) as well as streptavidin horseradish peroxidase (DAKO, Hamburg, Germany) and diaminobenzidine (Vector Labs, Burlingame, CA, USA), which yields a brown reaction product. Counterstaining was performed using Mayer’s hematoxylin.

BAFF and B‐cell (B220) detection by immunohistochemistry (IHC) was performed on formalin‐fixed, paraffin‐embedded mouse lung sections. Briefly, lung sections (3 μm) were deparaffinized in xylene and rehydrated in ethanol:water. Endogenous peroxidases were blocked in 3% H₂O₂ in methanol. Citrate buffer antigen retrieval was performed (45 min). Sections were blocked with 1% swine serum in Tris‐buffered saline (TBS) with 0.01% Tween 20. Sections were stained with a rabbit IgG anti‐mouse BAFF (1:50; Abcam) or a rabbit IgG anti‐mouse B220 (1:100, Abcam) at 4°C overnight. Primary antibody was detected with a biotinylated goat anti‐rabbit IgG (1:100; 1 h RT) followed by a 30 min incubation with streptavidin‐Horse Radish Peroxidase (Dako, Glostrup, Denmark). Staining was visualized using 3‐amino‐9‐ethylcarbazole (AEC). Pulmonary TLTs were assessed based on morphology as previously described (Morissette et al. 2014). Briefly, three photos at a 16x magnification were taken for every lung at two different cross‐sections. Whole lung cross‐section area (Lung_area [pixel²/10¹⁰]), as well as the number of bronchus‐associated TLT (TLT_number), were determined using Image J Software (https://imagej.nih.gov/ij/index.html) and identified according to their distinctive morphology. Data are expressed as bronchus‐associated TLTs per lung area (TLT_number/Lung_area).

Proliferating intestinal epithelial cells were stained with 5-Bromo-2’deoxyuridine (BrdU). At 90 minutes prior to sacrifice, mice received intraperitoneal injections of BrdU (5mg/ml in 0.9% saline, Sigma-Aldrich) to label S-phase cells [23 (link),24 (link)]. Jejunal tissue was then fixed in 10% formalin for 24 hours before being embedded in paraffin and slide-mounted in 5μm sections. Slides were then deparaffinized, rehydrated, and incubated in 1% hydrogen peroxide for 15 minutes before being heated in a pressure cooker in antigen decloaker (Biocare Medical, Concord, CA) for 45 minutes. Protein block (Dako, Carpinteria, CA) was performed for 30 minutes at room temperature and slides were incubated overnight at 4°C with rat monoclonal Anti-BrdU (1:500; Accurate Chemical and Scientific, Westbury, NJ). Samples were then incubated with goat anti-rat antibody (1:500; Accurate Chemical & Scientific) and streptavidin horseradish peroxidase (1:500; Dako), each for an hour at room temperature. Diaminobenzidine (DAB) was used to develop slides for 2–3 minutes, and counterstaining was performed with hematoxylin. BrdU-positive cells were quantified in 100 contiguous, well-oriented intestinal crypts.

Five-micrometer sections of decalcified femur were deparaffinized and rehydrated. After antigen retrieval, slices were washed in distilled water for 10 min at room temperature and immersed in increasing graded ethanol for 1 min. DNA was denatured at 95°C for 5 min, and hybridization process was performed with human Alu-DNA Probe (Biogenex, Fremont, CA, United States) at 37°C overnight. Hybridized slices were washed in standard saline citrate (pH 7.0; Dako) at room temperature for 10 min, 56°C for 10 min, and room temperature for 15 min. Nonspecific binding was prevented by incubation in PBS containing 3% bovine serum albumin (Sigma) for 1 min followed by a two-step avidin and biotin blocking (Vector Laboratories Ltd., Brussels, Belgium) for 15 min at room temperature. To detect hybridized Alu sequences, slices were incubated with biotinylated anti-fluorescent antibody (1/100) (Vector Laboratories Ltd.) for 1 h, and the signal was revealed using streptavidin/horseradish peroxidase (Dako) with diaminobenzidine as the chromogenic substrate.

Morbillivirus immunohistochemical (IHC) testing was conducted following an in-house developed diagnostic laboratory protocol, as previously described^{57 (link)}. Epitope retrieval was done using citrate buffer (pH 6.0). Nonspecific binding was blocked with 3% hydrogen peroxide and a commercially available blocking agent (Power Block, Biogenex, San Ramon, CA). The primary antibody was a mouse monoclonal antibody directed against the nucleoprotein of canine distemper virus (#CDV-NP, Veterinary Medical Research and Development, Pullman, WA) but known to cross-react with other morbilliviruses, at a final concentration of 1:400. The secondary antibody was biotinylated horse anti-mouse IgG, rat absorbed (Vector Labs, Burlingame, CA), and the conjugate Streptavidin–horseradish peroxidase (Dako, Carpinteria, CA). The substrate-chromogen system was 3,3′-diaminobenzidine (DAB) (Dako, Carpinteria, CA). Tissue sections were counterstained with Gill’s hematoxylin. Positive control was a CDV-infected formalin-fixed and paraffin embedded Vero cell pellet, while the negative control was similarly treated uninfected Vero cell pellet. Positive and negative controls were included in each IHC run.