Biotinylated donkey anti rabbit

The brains and the proximal portion of the small intestine were cut with a Leica CM3050S cryostat into 10 μm-thick serial sections in the coronal plane, collected and processed for immunofluorescence or immunoperoxidase with specific primary antibodies. The following primary antibodies were used: anti-occludin 1:200 (Abcam, Cat No. 222691; rabbit polyclonal), anti- Iba-1 1:1,000 (rabbit anti-ionized calcium binding adapter molecule 1; Wako Chemicals, Germany). The following secondary antibodies were used: biotinylated donkey anti-rabbit 1:100 (Vector Laboratories) for occludin and donkey anti-IgGs Alexa-488 (LifeTechnology). Slides were analyzed and images digitally acquired with a Leica DMI6000 microscope equipped with the Leica DFC320 cooled digital CCD camera (Leica Microsystems).

A free-floating protocol as described previously (Chlan-Fourney et al., 2011 (link)) was used. Briefly, the section was incubated in citrate buffer for antigen retrieval, blocked with 10% fetal bovine serum, and probed for 5-HTT (1:30 dilution; H-115 antibody). It was then incubated with biotinylated donkey anti-rabbit (1:200 dilution; Vector Laboratories Canada; Burlington, ON, Canada) and processed by the avidin–biotin–peroxidase/3,3′-diaminobenzidine (DAB) method. 5-HTT immunodetection in fixed-cells was done as described elsewhere (Allonby et al., 2014 (link)). Briefly, cells were plated on glass-bottom culture dishes, fixed in formaldehyde/PBS, permeabilized, and blocked in PBS containing normal horse serum (Sigma-Aldrich, Oakville, ON, Canada). Alexafluor-conjugated antibody was used for detection. ProLong™ Gold Antifade Mountant with DAPI (P36931) (Invitrogen; Burlington, ON, Canada) provided nuclear staining. Cells were visualized using 60X oil immersion on an Olympus FV1000 confocal microscope. Images were deconvoluted using Auto-Deblur (AutoQuant X3, Media Cybernetics) and processed with the Image J software.