Aec substrate chromogen

Immunohistochemistry was performed as described previously [18 (link)]. Briefly, after fixation of the cells with 4% formaldehyde, cells were permeabilized using 0,1% Triton X 100 in PBS for 10 min, and the endogen peroxidases were blocked with 3% H₂O₂ in methanol for 30 min. After three washing steps with PBS, the cells were incubated for 1 h with a 1:1000 dilution of primary antibody (SARS-CoV-2 (2019-nCoV) Nucleocapsid Antibody (Rabbit Mab, Sinobiological Cat: 40143-R019) in antibody diluent (REAL Antibody diluent, Agilent Technologies, Santa Clara, CA, USA, Dako Cat: S202230_2) followed by the treatment with the secondary antibody (EnVision™ + Dual Link System HRP, Agilent Technologies, Dako Cat: K5007). After washing (PBS 3x), the substrate (AEC substrate-Chromogen, Agilent Technologies, Dako, Cat: K346430-2, 2 drops) was dropped on the cells and incubated until viral infected cells were stained red. The reaction was then stopped with washing in PBS (3x) and wells were kept humid until photo documentation. For photo documentation, fourfold magnification with a Nikon Eclipse TS100 microscope (Nikon, Japan, Tokyo) was used with the JENOPTIK GRYPHAX^® camera and software (Jena, Germany) were used.

Tissue samples harvested at necropsy were placed in 10% neutral buffered formalin (Australian Biostain). Preserved tissue samples were processed according to routine histological methods before embedding in paraffin wax and sectioning into 4 µm-thick sections.
A rabbit anti-Influenzavirus A nucleoprotein hyperimmune serum (generated in-house by AAHL Bioassay R&D Team) was used as the primary antibody to detect viral antigen in formalin-fixed paraffin embedded tissues. Tissue sections were deparaffinized before high pH antigen retrieval (Dako, Agilent Technologies) at 97 °C for 30 min. Endogenous peroxidases were blocked with 3% hydrogen peroxide for 10 min. Tissue sections were incubated with a 1:2,000 dilution of the primary antibody for 60 min, washed with TBS prior to a 20 min incubation with anti-rabbit horseradish peroxidase-conjugated secondary antibody (Dako, Agilent Technologies). Viral antigen was visualized by the addition of AEC substrate chromogen (Dako, Agilent Technologies) for 10 min, washed with TBS and counterstaining with Mayer’s haematoxylin before visualising by light microscopy.
The presence of influenza nucleoprotein antigen in tissues was assessed by light microscopy. Antigen quantities were scored according to a four-point scoring system: 0, no antigen; 1, rare antigen; 2, moderate antigen; 3, abundant antigen.

PLP fixed mouse brains were paraffin-embedded and 5 μm tissue sections were cut and mounted on glass positive charge slides. Tissues were deparaffinized in a 65°C oven followed by successive xylene immersions (100%), rehydrated through graded ethanol washes (100% x 2, 95% x 2 and 70% at 5 min per wash), and 88% formic acid treated for 60 min. Tissues were then subjected to hydrated autoclaving using an automated antigen-retrieval system 2100-Retriever (Prestige Medical) and a citrate buffer (0.01M sodium citrate, 0.05% tween 20, pH 6) for 30 min. Samples were then blocked with 3% H₂O₂ (30 min) followed by a proprietary protein block (TNB, PerkinElmer Life and Analytical Sciences) (30 min) and stained with unconjugated BAR-224 at 2 μg/ml (Cayman Chemical) (overnight at 4°C). Detection was completed using HRP-conjugated anti-mouse secondary antibody (Envision+, Dako) (30 min) and AEC substrate chromogen (Dako) (1 min). Tissues were then counterstained with Meyer’s hematoxylin (Dako) (2 min), followed by 0.1% calcium bicarbonate bluing reagent (5 min) and coverslipped with aqueous mounting media (Dako).

To compare uNK numbers between the experimental groups, implantation sections were stained with DBA lectin. After dewaxing and rehydration procedure, endogenous peroxidases were blocked by incubation with 3% hydrogen peroxide (H₂O₂) in methanol at RT for 30 min in a humidity chamber. Slides were washed in 50 mM PBS twice, followed by 15 min avidin and biotin blocking each. Prior to DBA lectin staining [dilution 1:150 in 1% bovine serum albumin (BSA) in 100 mM PBS, overnight (ON) at 4°C], proteins were blocked with 1% BSA in 100 mM PBS for 30 min. For negative controls, DBA lection staining solution was replaced by 1% BSA in 100 mM PBS. At the next day, sections were washed twice with PBS and treated with horseradish peroxidase-solution for 30 min. After staining, slides were washed twice and incubated with AEC substrate chromogen (Dako) for 6 min and washed twice again. Sections were counter-stainined with hematoxylin solution for 1–2 min, dipping in warm tap water and mounted with Aqua Tex. uNKs were identified as DBA lectin reactive, brown cells, and their number was calculated per 1 mm² with the help of an eyepiece-micrometer (Zeiss) and a light microscope (Zeiss).

Formalin-fixed, paraffin-embedded cancer, and normal tissue samples from tissue array (BC081120d, PR1921c, CR1001a, and BC04002b, Biomax, Rockville, MD, USA) were analyzed by immunohistochemistry^{65 (link)}. Briefly, after de-paraffinization and rehydration, tissue sections were subjected to 1× Target Retrieval Solution, pH 6.0 (DAKO, Glostrup, Denmark) and then incubated with peroxidase blocking solution (DAKO) for 10 min at RT. They were then washed with 1× TBST buffer (Scytek Lab, Logan, UT, USA) followed by a protein block (0.25% casein in PBS, DAKO) for 10 min at RT and incubated overnight at 4 °C with primary antibodies. Sections were incubated with secondary antibodies for 1 h at RT after rinsing in TBST buffer. AEC substrate chromogen (DAKO) was added and washed with deionized water. The slides counter stained with Mayer’s hematoxylin (Sigma-Aldrich) were rinsed with tap water and mounted using an aqueous medium (Scytek Lab, USA). Ki-67 expression in xenograft tumors was determined as the percentage of positive cells per field and normalized by the total number of cells in each field.