Immpact amec red peroxidase substrate

MKP-2^+/+ and MKP-2^−/− EAE mice were sacrificed at the planned dates. Following PBS perfusion, intact spinal cords were flushed out with PBS by hydrostatic pressure using a sterile 19G needle. Frozen spinal cord tissue sections were then stained with standard haematoxylin and eosin (H&E) staining or with anti-CD45, anti-CD4, anti-CD8, anti-CD11b and anti-CD11c antibodies (all purchased from eBioscience) followed by incubation with an appropriate biotin-conjugated secondary antibody (eBioscience, UK), horseradish peroxidise and ImmPACT AMEC red peroxidase substrate (Vector Lab, UK) for detection. Isotypes with matching IgG were used as negative controls for all antibody staining.

Primary tumor samples were processed as previously described [34 ]. Briefly, primary tumor samples were fixed in 4% paraformaldehyde-PBS overnight, and then transferred to 30% sucrose until tissue samples sank (2–3 d). Tissue samples were transferred to cryomolds and embedded in optimal-cutting temperature (OCT) compound (TissueTek). 10 μm cryosections were generated on a cryostat (Leica). Endogenous peroxidase activity was neutralized (Bloxall, Vector Laboratories) before samples were permeabilized (0.5% Triton X-100, TBS) and blocked (5% horse serum, Vector). Immunohistochemical labeling was performed for CD3 (Biolegend, 300302, 3 h RT). CD3 was developed with a peroxidase secondary (ImmPRESS VR anti-rabbit IgG, Vector) and ImmPACT AMEC Red Peroxidase Substrate (Vector). Tissue samples were counterstained with Hematoxylin QS (Vector, 45 s, RT), developed in a bluing solution (0.1% NaCO₃/H₂O, 1 min) and coverslipped with Fluoro-Gel with Tris buffer (Electron Microscopy Services). Samples were imaged on a Nikon Eclipse.

Formalin-fixed, paraffin embedded tissues were sectioned into 3 μm and stained with hematoxylin-eosin or used for immunohistochemical staining (IHC). IHC was performed using YEZV-N peptide (POS: 165–182) immunized rabbit serum (1:20,000) (Cosmo Bio, Tokyo, Japan) or anti Iba1 goat polyclonal antibody (1:500, #001–27991, Wako, Osaka, Japan) as primary antibodies. For YEZV antigen detection, peroxidase-labeled goat anti-rabbit IgG polyclonal antibody (Nichirei Bioscience, Tokyo, Japan) and DAB were used as secondary antibodies and substrates, respectively. In the Iba1 and YEZV antigen double staining experiments, ImmPRESS (Peroxidase) Polymer Anti-Goat IgG Reagent (Vector laboratories, California, USA) and alkaline phosphatase-labeled goat anti-rabbit IgG polyclonal antibody (Nichirei Bioscience, Tokyo, Japan) were used as secondary antibodies and ImmPACT AMEC Red Peroxidase Substrate (Vector laboratories, California, USA) and Vector Blue (Vector laboratories, California, USA) as substrates. All images were acquired using Nikon ECLIPSE 80i light microscopy (Nikon, Tokyo, Japan).