Immunohistochemistry for SARS-CoV-2 Nucleocapsid Protein

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.