Cd3 ab5690

Specimens of excised livers and spleens were paraffin embedded and sectioned at 4 µm thickness. For histological assessment, deparaffinized liver sections were stained with hematoxylin (Merck, Darmstadt, Germany) and eosin Y (Carl Roth, Karlsruhe, Germany). For immunohistochemistry, deparaffinized organ sections were boiler-treated in EnVision™ FLEX Target Retrieval Solution, High pH (DAKO, Glostrup, Denmark) or 10 mM sodium citrate buffer pH 6.0 (applies to the NKp46 antibody only) for 30 min, blocked with 10% goat or 2.5% horse serum, incubated with the primary antibody overnight at 4°C (CD68: ab125212; CD3: ab5690; NKp46: ab214468 – all Abcam, Cambridge, UK), treated with 3% hydrogen peroxide and incubated with a biotinylated anti-rabbit secondary antibody (Vector Laboratories, Burlingame, CA, USA) at a dilution of 1:500. Peroxidase activity was developed with an avidin-biotin-enzyme complex (ABC) (Vector Laboratories) and AEC+ chromogen (DAKO), nuclei counterstained with hematoxylin, and sections mounted with aqueous mounting media, Aquatex® (Merck). Mounted sections were visualized using a Scope A.1 microscope and photomicrographed with an AxioCam MRC camera (Carl Zeiss Microscopy, Jena Germany). For quantification of NK cells, positively stained cells in sections treated with the NKp46 antibody were manually counted and calculated per mm² of liver tissue.

Immunohistochemistry was adapted from the manufacturer’s protocol (PK-6101, Vectastain Elite ABC kit peroxidase, Vector Laboratories) and⁴⁶ (link) with the following modifications: 5 μm slices of paraffin embedded liver were rehydrated and processed for antigen retrieval with citrate buffer pH6 (C999, Sigma Aldrich) or tris-EDTA buffer pH9. Then, endogenous peroxidase activity was quenched with 3% hydrogen peroxide (H1009, Sigma Aldrich) and non-specific binding was prevented with an avidin/biotin blocking kit (SP-2001, Vector Laboratories) and 5% goat serum (Vector Laboratories). Afterwards, primary rabbit antibodies (CD68 ab12512; myeloperoxidase MPO ab208670; CD3 ab5690, Abcam) and secondary biotinylated goat anti rabbit antibodies (Vector Laboratories) were used. After incubations and washes with PBS, colorimetric substrate was added (E109, HistoGreen, Novus Biological) and slices were counterstained with Mayer hematoxylin (51275, Sigma Aldrich). Vectamount permanent medium (Vectorlab) was used to glue the coverslips on the slides. Images were acquired using an Axio Scan microscope (Zeiss) and Zen® software (Zeiss).