In the first step, tonsillar tissue sections wereimmunostained with 15 different types of lymphocyte markers torecognize B cells, T cells, natural killer cells, and plasma cells,such as antibodies against CD3 (PS1; mouse IgG2a), CD10 (56C6;mouse IgG1; both Nichirei, Tokyo, Japan), CD20 (L26; mouse IgG2κ;Dako), CD23 (1B12; mouse IgG1κ), CD38 (SPC32; mouse IgG1; bothNovocastra), CD56 (1B6; mouse IgG1), CD79a (JCB117; mouse IgG1κ),CD117 (Rabbit polyclonal; all Nichirei) CD138 (MI15; mouse IgG1κ),IRF4/MUM-1 (MUM-1p; mouse IgG1κ; both Dako), BCL6 (rabbitpolyclonal; Santa Cruz Biotechnology, Inc., Dallas, TX, USA),paired box protein 5 (PAX5; 8F9; mouse IgG1κ, Abgent, San Diego,CA, USA), programmed cell death 1 (PD1)/CD279 (NAT; mouse IgG1;Abcam, Cambridge, UK), BLIMP1 (3H2-E8; mouse IgG1; Thermo,Rockford, IL, USA), and transmembrane activator and calcium-signalmodulating cyclophilin ligand interactor (TACI; 4976; mouse IgG,Santa Cruz Biotechnology, Inc.). A positive reaction was detectedwith the UltraTech HRP Streptavidin-Biotin Detection System(Beckman Coulter, Marseille, France) and DAB as a brown color.Digital photomicrographs were taken of high-power-view fields inthe SYM/MZ and light zone, which contained a large number ofTRPM8+ cells. In the second step, TRPM8 immunostainingwas performed on the same tissue sections using the same method,and digital photomicrographs of the same fields as those in thefirst step were taken. Photos, bilayered by TRPM8 singleimmunostaining and TRPM8-lymphocyte marker double immunostaining,were created using image processing software (Adobe PhotoshopElements 7.0, Adobe Systems, San Jose, CA, USA)(33). TRPM8 single-positive cellsand double-positive cells in each of the 5 imaging photos of theSYM/MZ and light zone were counted and the ratio of double-positivecells to TRPM8+ cells was calculated.
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