Goat anti mouse igg2a fitc

Staining was performed on 5 µm frozen tissue sections. Mouse anti-CD20 (Dako) was developed with goat anti-mouse IgG2a FITC (Southern Biotech), rabbit anti-RANKL (AbCam) was developed with donkey anti-rabbit Rhodamine (Jackson ImmunoResearch), and mouse anti-FcRL4 (BioLegend) was developed with goat anti-mouse IgG2b Cy5 (Southern Biotech). Isotype-matched irrelevant controls were used (Jackson ImmunoResearch). Sections were incubated with primary antibody for 1 h and secondary antibody for 30 min. Sections were mounted using a DAPI-containing mounting medium. Sections were visualised using a Zeiss confocal LSM 510 microscope and images were processed using Zeiss LSM Image Examiner software.

Lungs were fixed for 24 h with 10% formalin and were kept inflated at constant pressure throughout the fixation process. Lung sections were stained with hematoxylin and eosin, and were evaluated blindly by two experienced lung pathologists. Images were taken with a Leica DFC295 microscope. Immunofluorescence staining was performed on 4 μm acetone-fixed frozen lung and kidney sections. Tissue sections were incubated with the primary anti-α-SMA (Cat. Nr: 61001, ProGen, Heidelberg, Germany) antibody for 1 h at RT. Binding of α-SMA primary antibody was detected by incubation with goat anti-mouse IgG2a FITC (Southern Biotech, Birmingham, AL, USA) for 30 min. Nuclei were stained with Hoechst, and sections were coverslipped with Prolong Gold antifade reagent (Invitrogen). All immunofluorescence images were taken with a Zeiss Axiovert 200M inverted microscope and AxioVision acquisition software (Carl Zeiss, Oberkochen, Germany).