Anti il 34 antibody

Immunofluorescent staining was performed on Caco-2 cells cultured on chamber slides (Sarstedt) that were fixed in acetone followed by blocking with 10% (v/v) normal goat serum in PBS. Anti-IL-34 antibody (catalogue number AB-75723; Abcam) was added, and slides were incubated overnight at 4°C. chamber slides were incubated with secondary goat anti-rabbit IgG conjugated with Alexa Fluor® 488 (catalogue number A11034; Invitrogen), diluted in blocking buffer. Sections stained with the secondary antibody alone were used as negative controls. Sections were mounted in ProLong Gold antifade reagent with DAPI (Invitrogen). Slides were scanned on a LSM710 confocal microscope (Zeiss) in single-photon mode.

Intestinal biopsies were immediately placed in Histocon solution (Histolab Products) after excision, embedded in optimal cutting temperature compound (OCT; Histolab Products), and kept at −80°C until sectioning. Sections of 7 μm were subjected to fixation in acetone, blocking of endogen peroxidase in methanol and H₂O₂ followed by blocking of endogenous alkaline phosphatase by 20% acetic acid. Sections were blocked using avidin and biotin solutions (Vector Laboratories) and incubated in appropriate serum. Anti-IL-34 antibody (catalogue number AB-75723; Abcam) was added, and slides were incubated overnight at 4°C. Sections were incubated with the secondary antibodies using goat anti-rabbit (biotinylated) (catalogue number BA-1000; Vector Laboratories), followed by incubation in Vectastain ABC complex (Vector Laboratories), and development in DAB (diaminobenzidine) solution (Vector Laboratories). Sections were dehydrated and mounted in histograde mounting media (Histolab Products). Rabbit IgG control (catalogue number I-1000; Vector Laboratories) was used as isotype control.