Anti rabbit ig peroxidase

To detect any type of mCMV-infected cells, one-color IHC specific for the intranuclear viral protein IE1 was performed on liver tissue sections as described in detail elsewhere [107 ]. For differentiating infected cells by cell type, 2C-IHCs were performed by combining IE1-specific labeling with the labeling of cell type-specific markers, such as CD31 for the identification of endothelial cells (EC) or F4/80 (Ly71) antigen for the identification of macrophages (MΦ), essentially as described recently for a 3C-IHC analysis that has included the same markers [36 (link)]. 2C-IHC specific for IE1 and active caspase 3 was described previously [114 (link)] and applied with modifications. In brief, intranuclear IE1 protein was labeled with monoclonal Ab CROMA 101 and stained in red with alkaline phosphatase-1-conjugated polyclonal goat anti-mouse IgG (AbD Serotec, Puchheim, Germany) and fuchsin substrate-chromogen kit 2 (Dako-Cytomation, Hamburg, Germany). Active caspase 3 was detected with rabbit anti-active caspase 3 IgG (Biovision, Milpitas, CA, USA) and stained in brown by using the ImmPRESS reagent: anti-rabbit-Ig-peroxidase (Vector Laboratories, Peterborough, UK) with 3,3’-diaminobenzidine (DAB) as substrate. A light blue counterstaining was achieved with hematoxylin.

Staining was performed on 5 µm frozen acetone-fixed sections. Endogenous peroxidase activity was blocked with Bloxall Solution (Vector) and 2% casein was used to block non-specific staining. Sections were washed with phosphate buffered saline (PBS) between incubation steps. Sections were incubated with anti-CD20 (Dako), mouse anti-FcRL4 (Biolegend), or isotype-matched irrelevant controls (Jackson ImmunoResearch) for 1 h, rabbit anti-mouse Ig (Dako) for 10 min, and anti-rabbit Ig peroxidase (Vector Laboratories) for 30 min. Staining was developed using a diaminobenzidine substrate (Vector Laboratories). Slides were counterstained with haematoxylin and mounted with dibutyl phthalate xylene (DPX) (Sigma Aldrich).