Alexa fluor 555 conjugated anti rabbit igg

The utilized primary antibodies included mouse monoclonal anti-human insulin and rabbit polyclonal anti-human c-peptide antibodies (Cell Signaling, Denver, USA). The employed secondary antibodies were Alexa Fluor 488-conjugated anti-mouse IgG (H+L) and Alexa Fluor 555-conjugated anti-rabbit IgG (Cell Signaling). The cells were fixed in 4% paraformaldehyde, permeabilized with chilled 100% methanol for 10 min, blocked with 5% normal goat serum for 60 min at RT and incubated with the primary antibodies overnight at 4°C. The cells were then washed with PBS and incubated with the secondary antibodies for 2 hours.

Three μm cryosections were fixed in −20°C methanol for 10 min, washed with 7.4% PBS 3 times, treated with Protein Block Serum-Free for 30 min, and washed with PBS 3 times. Then, the samples were incubated with mouse monoclonal anti-claudin 1 antibody (1 : 100) (ab56417, Abcam) for 2 h at room temperature, washed with PBS 3 times, and incubated with Alexa Fluor 488 conjugated anti-mouse IgG (1 : 1000) (Cell Signaling Technology, Inc., Danvers, MA, USA) for 30 min. Again, the samples were washed with PBS 3 times, treated with Protein Block Serum-Free for 30 min, washed with PBS 3 times, and incubated with rabbit polyclonal anti-ZO-1 (1 : 400) (Invitrogen) at 4°C overnight. Samples were washed with PBS 3 times, and incubated with Alexa Fluor 555 conjugated anti-rabbit IgG (1 : 1000) (Cell Signaling Technology) for 30 min. Nuclear staining was performed using DAPI. Immunostaining for occludin was also performed using rabbit polyclonal anti-occludin antibody (Cat. 71-1500, Invitrogen, US) (1 : 200) and Alexa Fluor 555 conjugated anti-rabbit IgG. As a negative control, PBS was used instead of the primary antibodies. Immunofluorescence was examined with a laser scanning confocal microscope (OLYMPUS BX-51, Olympus, Tokyo, Japan).