Immunohistochemistry (IHC)
A polyclonal antibody against Mycobacterium bovis (Bacillus Calmette‐Guerìn, BCG, code no. B 0124 Dako, Denmark), that was previously applied in a study about etiological deepening study of granuloma in Doctor fish (Garra rufa ) (Volpe et al., 2019) was employed. Endogenous peroxidase inhibition was made with 3% H2O2 methanol solution. The antigen retrieval was made by using a microwave oven at 750W, 2 cycles × 5min. Preincubation with a blocking solution (10% Normal Goat Serum and Phosphate Buffered Saline solutions) was performed for 30min and then incubated overnight at 4°C with a primary antibody diluted at 1:3,000 in a blocking solution. The secondary anti‐rabbit antibody was incubated at 1:200 dilution for 30min, followed by a revelation system with an Avidin-Biotin Complex (ABC) kit developed with diaminobenzidine (DAB) chromogen for 90s and counterstained with Papanicolaou haematoxylin.
To study Cutibacterium acnes (formerlyPropionibacterium acnes ), polarized slides were sent to Department of Human Pathology, Tokyo Medical and Dental University, for IHC with a C. acnes -specific monoclonal (PAB) antibody that reacts with lipoteichoic acid, the cell membrane constituent of the bacterium, in formalin-fixed and paraffin-embedded (FFPE) tissues. IHC with a commercially available PAB antibody (#D371-3, MBL, Nagoya, Japan) was performed by Leica BOND-III (Leica Microsystems Inc., Tokyo, Japan) using a BOND Polymer Refine Detection kit (#DS9800, Leica Microsystems Inc.) as described in the study by Isshiki et al., 2021. Deparaffinization, peroxidase inhibition, antigen retrieval with BOND Epitope Retrieval Solution 1 (#AR9961, Leica Microsystems Inc.) at 100 ◦C for 60 min, incubation with PAB antibody (diluted 1:500) at room temperature for 8 min, and counterstaining with Mayer’s hematoxylin were performed according to the manufacturer’s protocol.
Positive internal and negative control slides were processed in parallel by replacing the primary antibody with a non-reactive isotype-matched antibody.