Alexa fluor 555 conjugated anti rabbit igg

For immunofluorescence staining, cells on slides were fixed with 4% paraformaldehyde for 10 min, then washed with PBS for three times and blocked in blocking buffer (1% BSA, 0.3% Triton X-100, and 10% goat serum in PBS) for 1 h, followed by the incubation with anti-TH at 4°C for overnight. After washing with carrier buffer (1% BSA, 0.3% Triton X-100, and 1% goat serum in PBS), they were incubated with Alexa Fluor 555-conjugated anti-rabbit IgG (#A0453, Beyotime) for 2 h at room temperature, followed by DAPI staining for 5 min. Then, cell slides were mounted for taking pictures. Immunochemistry staining was performed according to the manufacturer's protocol (#95-6143, Invitrogen). In brief, free-floating 25 μm-thick serial sections were tightly attached to the microslides and then were treated with 0.3% hydrogen peroxide for 10 min followed by incubation with anti-TH at 4°C for overnight. After washing with PBS, the sections were incubated with a biotinylated second antibody (Reagent 1B) followed by the conjugate enzyme (Reagent 2) for each 10 min. Finally, a chromogen AEC single solution was used to develop the signals, and pictures were captured on a microscope (BX51TF, Olympus, Tokyo, Japan).

Uterus were collected from mice after cardiac perfusion with PBS and fixed in 4% formaldehyde solution immediately and made into paraffin sections. Paraffin sections were stained with hematoxylin and eosin (HE). Paraffin sections of uterus were rehydrated and boiled in EDTA buffer (pH 6.0) for 20 min to induce antigen retrieval. After washing, tissue sections were blocked with 5% goat serum for 30 min at 37°C, followed by staining with rabbit anti-mouse CD3 antibody (Abcam, polyclone, 1:100) and armenian hamster anti-mouse γδT antibody (Santa Cruz Biotechnology, clone: UC7-13D5, 1:100) at 4°C overnight. Sections were washed four times for 5 min each time and incubated with Alexa Fluor 555-conjugated anti- rabbit IgG (Beyotime, 1:1000) plus DyLight 488-conjugated anti-Syrian hamster IgG (Abcam, 1:1000) for 30 min at 37°C in the dark. After a final washing, cover slips were mounted onto slides with fluoroshield mounting medium with DAPI (4, 6-diamidino-2-phenylindole, Abcam). Images were captured with Olympus microscope BX53 and processed with LSM Image Examiner software (Zeiss).

Paraffin sections of lung tissues from wild type (WT) and infected mice were rehydrated and boiled in Sodium citrate buffer (pH 6.0) for 30 min to induce antigen retrieval. After washing, tissue sections were blocked with 10% goat serum, followed by staining with rabbit anti-mouse CD3 antibody (Abcam) and hamster anti-mouse γδT antibody (Santa Cruz Biotechnology) at 4°C overnight. Sections were washed and incubated with Alexa Fluor 555–conjugated anti-rabbit IgG plus Alexa Fluor 488–conjugated anti-hamster IgG (Beyotime, Shanghai, China) for 30 min at 37°C in the dark. After a final washing, cover slips were mounted onto slides with fluoroshield mounting medium with DAPI (Abcam). Images were captured with Olympus microscope BX53 and processed with LSM Image Examiner software (Zeiss).