3 3 diaminobenzidine tetrahydrochloride dab

A detailed protocol is provided in the supplementary methods. Briefly, 5 µm tissue sections were de-paraffinized in xylene and rehydrated using decreasing alcohol concentrations. Antigen retrieval and endogenous peroxidase block were performed in citric acid buffer (10 mM citric acid, pH 6, 0.1% Tween 20) and 3% H₂O₂ in PBS, respectively. Samples were then incubated in blocking solution (5% bovine serum albumin (BSA, Merk) and 1% donkey serum (Dianova GmbH) in PBS). Primary and secondary antibodies (see supplementary Table S3-4) were diluted in blocking solution and incubated in a dark humidified chamber. Biotinylated secondary antibodies (GE Healthcare, see supplementary Table S4) and ExtrAvidin-Peroxidase (Sigma-Aldrich) were diluted in PBS, and samples were incubated in a dark humid chamber. Staining was developed using 3,3′-diaminobenzidine-tetrahydrochloride (DAB; Roth) and counterstained using hematoxylin. Slides were dehydrated following the reverse order of the alcohol gradient and mounted with Histokitt (Carl Roth GmbH).

Immunohistochemistry was performed to detect SARS-CoV-2 antigen (SARS-CoV-2 nucleoprotein), macrophages and dendritic cells (ionized calcium-binding adapter molecule 1, IBA-1), alveolar pneumocytes type 2 (pro-surfactant protein C), alveolar differentiation intermediate cells (cytokeratin 8), airway basal cells (cytokeratin 14), club cells (secretoglobin 1A1), and M2 macrophages (CD 204). Immunolabelings were visualized either using the Dako EnVision+ polymer system (Dako Agilent Pathology Solutions) and 3,3´-Diaminobenzidine tetrahydrochloride (DAB, Carl Roth) as previously described^{36 (link)} or using avidin–biotin complex (ABC) peroxidase kit (Vector Labs) and DAB (Carl Roth) as previously described^{74 (link)}. Nuclei were counterstained with hematoxylin. Further details about primary and secondary antibodies, visualization methods and dilutions used can be found in Supplementary Table 2. For negative controls, the primary antibodies were replaced with rabbit serum or BALB/cJ mouse ascitic fluid, respectively, with the dilution chosen according to protein concentration of the exchanged primary antibody. Antibodies were tested on murine and human lung tissue to confirm specificity for the cells of interest. Subsequently, murine and human tissues were used as positive controls.