Rabbit anti cd45 antibody

Flow cytometry was performed on lymphocytes isolated from the above-mentioned tissues. All tissues were finely cut, transferred into PBS containing 1 mg/mL collagenase (034-22363, Wako, Osaka, Japan), and incubated at 37 °C for 10 min in a shaking water bath. The digested tissues were washed with PBS and filtered through a 40-μm stainless steel mesh to separate the cells from the excess tissue debris. To detect leukocytes, 1 × 10⁶ leukocytes were incubated with rabbit anti-CD45 antibody (1:200; Abcam, Cambridge, MA, USA) for 60 min, rinsed, and then incubated with Alexa Fluor 647 donkey anti-rabbit IgG antibody (1:200; Molecular Probes, Leiden, The Netherlands) for 1 h. Finally, the cell suspensions were fixed in 500 μL of 1% formalin in PBS. The number and percentage of FITC⁺/CD45⁺ mononuclear cells in tissue was analysed on a Gallios™ flow cytometer (Beckman Coulter Inc., Fullerton, CA, USA) based on forward and side light scattering properties (Fig. 1c). A total of 10⁴ events per sample were collected, and positive cells were expressed as a proportion of mononuclear cells. Rabbit IgG isotype controls (Abcam) were used to detect non-specific staining and establish the criteria for positive cell populations (data not shown).