As read-outs, an immunohistochemical technique was performed in order to identify the immunoexpression of IL-33 (anti-IL-33, Polyclonal/rabbit, code A8096, 1:800 dilution, ABclonal, Manhattan Beach, CA, USA), B1R (anti-B1R, Polyclonal/rabbit, GTX70845, 1:100, GeneTex, Irvine, CA, USA), B2R (anti-B2R, Polyclonal/rabbit, ab236093, 1:100, Abcam, Cambridge, UK), CASP-1 (anti-CASP-1, Polyclonal/rabbit, ab189796, 1:200, Abcam, Cambridge, UK) and ACE2 (anti-ACE2, Polyclonal/rabbit, ab272690, 1:50, Abcam, Cambridge, UK) for observation of its immunoexpression in alveolar macrophages, endothelial cells and, type-I and -II pneumocytes. Tissue immunoexpression of Immunoglobulin (Ig) E (anti-IgE, Polyclonal/rabbit, BSB3070, 1:100, Bio SB, Santa Barbara, CA, USA) was used to quantify IgE+ MCs. Immunoexpression of tryptase (anti-Tryptase, Monoclonal/rabbit, EP259, 1:400, BioSB, Santa Barbara, CA, USA) was used to identify activated Tryptase+ MCs, as well as MCs in the process of degranulation of this enzyme.
The secondary polymer was the multipurpose developer’s Mouse and Rabbit Specific HRP/DAB IHC Detection Kit - Micro-polymer, ab236466 (Abcam, Cambridge, UK). Specificity controls were performed by (i) omitting the primary antibody (negative control) and (ii) conducting a tissue sample test on positive controls for each immune marker.
The secondary polymer was the multipurpose developer’s Mouse and Rabbit Specific HRP/DAB IHC Detection Kit - Micro-polymer, ab236466 (Abcam, Cambridge, UK). Specificity controls were performed by (i) omitting the primary antibody (negative control) and (ii) conducting a tissue sample test on positive controls for each immune marker.