Mom peroxidase kit

We obtained paraffin sections from TRAMP mice with PCa treated with BA (10 mg/kg body weight) or vehicle control from our previous study^{8 (link)}. Immunostaining for Ub-H2A (D27C4) (Cell Signaling) was performed using a 1/200 dilution of mouse monoclonal and the MOM Peroxidase Kit (Vector Laboratories) following the manufacturer’s instructions and as we previously described^{68 (link)}.

Five-week-old female BALB/c mice (Japan SLC), under mixed anesthesia (medetomidine-butorphanol-midazolam), were intranasally inoculated with 50-μl samples of 1 to 10⁵ FFU virus in PBS. The body weights and survival of the mice were monitored daily for 14 days. Mice that lost more than 30% of their original weight were humanely euthanized. Lungs and brains of mice infected with 10 FFU virus were collected 3 and 6 dpi, and virus titers were assayed by focus-forming assays. Enzyme-linked immunosorbent assays (ELISAs) were performed on lung homogenates using CCL2 (MCP1), IL-6, IL-10, TNF-α, IFN-γ, and IFN-β Quantikine kits (R&D Systems). For histopathology analysis, the lungs of mice infected with 10 FFU of virus were collected at 3 dpi, fixed in 4% buffered paraformaldehyde, embedded in paraffin, cut into 5-μm sections, stained with hematoxylin and eosin, and examined by light microscopy. Immunohistochemical staining of the viral antigen was performed, as described previously (17 (link), 41 (link)), using a monoclonal antibody (C43) specific for influenza A virus nucleoprotein and a Mouse-on-Mouse (MOM) peroxidase kit (Vector Laboratories) with diaminobenzidine as the chromogen and hematoxylin as the counterstain. Unrelated antibodies were used in place of the primary antibody as controls.

Sections of S. mansoni-infected or uninfected paraffin-embedded mouse tissues [liver and intestine/ileum from random-bred CD1 mice (Charles River, UK)] were stained for basophils with the basophil-specific rat anti-mMCP-8 antibody (clone TUG8, Biolegend), either by immunofluorescence or immunohistochemically. For immunofluorescence, anti-mMCP-8 was diluted 1:500. Binding was detected by AF-633-labeled secondary rabbit anti-rat IgG (Invitrogen), dilution 1:1000. Nuclei were counterstained by DAPI (Invitrogen; dilution 1:1,000), and pictures were taken with an Olympus IX-81 inverse fluorescence microscope (Olympus). Sections of S. mansoni-infected paraffin-embedded mouse liver tissue from female C57BL/6-NTAC mice (Taconic, Denmark) were immunohistochemically stained the anti-mMCP-8 and the secondary biotinylated rabbit anti-rat antibody were diluted 1:100, and detection was performed using the Vectastain Kit (Vector Laboratories). For detection of IPSE/alpha-1 the monoclonal antibody anti-IPSE/alpha-1 (clone 74 1G2) (16 (link)), was biotinylated according to standard protocols and diluted 1:100. To prevent non-specific binding to the mouse tissue, the M.O.M. Peroxidase kit (Vector laboratories) was applied. Mayer‘s hematoxylin (Merck) was used for counter staining. Pictures were taken at the Olympus BX51 microscope.