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.