Sigmafast 3 3 diaminobenzidine substrate

Peroxidase-linked assay (PLA) was performed as described previously [112 ]. A monolayer of MDCK cells on 96-well plates was inoculated with A/equi/Kentucky/81 H3N8 (1 h, 10 MOI). After 24 h of incubation at 37 °C, cells were washed, fixed, and incubated with serum from a rabbit immunized with A/equi/Kentucky/81 (HI titer 8024), horseradish peroxidase conjugate and SIGMAFAST™ 3,3′-diaminobenzidine substrate (Sigma-Aldrich, St. Louis, MO, USA). The results were analyzed under a microscope Nikon eclipse TS100 at a magnification of 40×, and pictures were taken with a Nikon D5000.

Mouse femur and tibia were fixed in 10% neutral-buffered formalin, decalcified in 12% EDTA and embedded in paraffin. Sections (5 μm) were stained with hemotoxylin & eosin to evaluate morphology. For 4-HNE immunohistochemistry, bone sections were de-paraffinized, heated in antigen unmasking solution (Dako, Carpinteria, CA) and permeabilized in phosphate-buffered saline (PBS, pH 7.4) containing 0.2% Triton-X-100. Sections were blocked in 1% bovine serum albumin (BSA) for 1 h, incubated with primary antibody (Alpha Diagnostics, San Antonio, TX) overnight at 4°C, followed by incubation in peroxidase-conjugated antibody (Vectastain Elite Reagent, Vector Labs, Burlingame, CA) and in Sigma Fast 3,3′-diaminobenzidine substrate (Sigma, St. Louis, MO). Sections were counterstained with 0.1% Methyl Green in PBS and examined by light microscopy. For quantification, three sections per mouse (n = 3/group) were immunostained twice and analyzed using Image J. Briefly, channels were separated, the blue channel image was inverted and the threshold adjusted to remove nuclear background. Average staining intensity and stained cell counts were measured in several ROI of the same size.