Diaminobenzidine dab substrate

IHC staining was performed as previously reported [29 (link)] with minor modifications. Briefly, 3 μm thick formalin-fixed, paraffin-embedded tissue sections were deparaffinized with xylene for 10 min, rehydrated through a gradient of 100, 95, 80, and 70% ethanol, and equilibrated in Tris buffer (TBS; 0.1 M Tris-HCl, pH 7.4 and 0.15 M NaCl). Antigen retrieval was performed by placing the slides in 0.01 M citric buffer (pH 6.0) and autoclaving at 121 °C for 20 min. The slides were treated with a protein-blocking agent for 10 min to reduce the non-specific binding of antibodies to the tissue and subsequently washed twice. Primary monoclonal antibodies anti-SV40 LT (mia90661; Thermo Fisher, Vilnius, Lithuania) and anti-JCPyV capsid VP1 (ab34756; Abcam, Cambridge, USA) were used to detect the expression of the early viral protein LT and the late structural protein VP1, respectively. Tissue sections were incubated overnight in a humidified chamber at 37 °C, followed by incubation after the sequential addition of biotinylated secondary antibody (PK6102, Vectastain ABC kit, Burlingame, CA, USA) and avidin-biotin complex (Vectastain ABC kit, Burlingame, CA, USA) for 1 h each. After development in the presence of diaminobenzidine (DAB) substrate (Sigma–Aldrich, St. Louis, MO, USA), sections were counterstained using hematoxylin.

Sections embedded in methyl methacrylate were deplasticized in methyl glycol acetate. After rehydration in a graded ethanol series to pure distilled water, non-specific peroxidases were blocked for 15 min with ortho-periodic acid and background activity was blocked at room temperature using 5% bovine serum albumin (BSA). Sections were then incubated in a humid atmosphere for 12 h at room temperature in a dark chamber with primary antibody against sclerostin (SOST) (R&D Systems, Minneapolis, MN, United States) diluted at 5 μg/mL. Sections were washed and then incubated with polyclonal antigoat immunoglobulin (Dakocytomation) diluted at 1/200 for 1 h at room temperature in a dark chamber. Peroxidase activity was detected using diaminobenzidine (DAB) substrate (Sigma–Aldrich). Control incubations to assess non-specific staining consisted of the same procedure except that the primary antibody was replaced by non-immune serum.

Lung samples were fixed in 4% paraformaldehyde overnight, embedded in OCT compound or paraffin and sectioned. For immunohistochemistry, the sections were blocked in 1% bovine serum albumin (BSA) (Jackson ImmunoResearch) and incubated with primary antibodies. The antibodies used were FITC mouse anti-β-catenin (1:400, BD Transduction Laboratories) and anti-α-SMA (1:200, Sigma). For whole mount PECAM-1 staining, fixed lungs were dehydrated in methanol, bleached in 3% hydrogen peroxide in in methanol, rehydrated, blocked in 1% BSA, incubated with anti-PECAM-1 antibody (1:200, Santa Cruz, sc-1506) overnight at 4°C, washed in PBS, incubated with HRP-conjugated anti-goat IgG (Santa Cruz) and colorized with diaminobenzidine (DAB) substrate (Sigma).