Anti mouse cd3

Lung tissue sections were deparaffinized in xylenes and were rehydrated by passage through serial dilutions of ethanol in distilled water. Heat-induced antigen retrieval was performed in an antigen retrieval solution (ImmuoActive; Matsunami Glass Ind., Ltd., Kishiwada, Japan) with the use of a microwave, twice, for 5 min. The sections were incubated with Blocking One Histo (Nacalai Tesque) in order to block non-specific reactions. Anti-mouse B220 (eBioscience), anti-mouse CD3 (Cell Signaling Technology) monoclonal antibodies (mAbs), and anti-mouse CD23 (Boster Biological Technology) polyclonal Ab were applied to the sections overnight, at 4°C. After washing with phosphate-buffered saline (PBS), the sections were incubated with an Alexa Fluor 488-conjugated anti-rat IgG (Invitrogen) or an Alexa Fluor 568-conjugated anti-rabbit IgG (Invitrogen) Ab. After washing thrice with PBS, the nuclear DNA was stained with 4′,6-diamidino-2-phenylindole (DAPI; Invitrogen, Waltham, MA), and the sections were observed under an optical microscope (ZEISS Axio Observer 7; Carl Zeiss) and were analyzed through the use of ZEN3.2 blue edition (Carl Zeiss).

Deparaffinized sections from lung tissues were used. Heat-induced antigen retrieval was performed in ImmunoActive, and the sections were incubated with Blocking One Histo. Subsequently, the sections were incubated with anti-mouse CD3 (Cell Signaling Technology), CD19 (Cell Signaling Technology, Danvers, MA), F4/80 (Cell Signaling Technology), CD11b (Cell Signaling Technology), or CD23 (Boster Biological Technology) Abs overnight, at 4°C. After washing with PBS, the sections were incubated with a horseradish peroxidase (HRP)-conjugated secondary Ab (Cell Signaling Technology). HRP reacted with the 3,3′-diaminobenzidine (DAB) substrate through the use of SignalStain DAB Substrate Kit (Cell Signaling Technology), and the sections were then counterstained with hematoxylin.