Cy3 conjugated goat anti rabbit immunoglobulin g

Six µm-thick cryostat cut muscle sections were mounted on polylysine-coated glass slides (Bio-Optica), air-dried for 30 minutes and stored at −80°C pending use. Sections (4 JDM, 4 DM, 4 PM, 4 sIBM, 4 maltase deficiency patients, and 4 controls) were fixed for 10 minutes with 4% paraformaldehyde, permeabilized for 5 minutes with ice cold methyl alcohol, and incubated with 10% normal goat serum diluted in PBS for 30 minutes at room temperature to block non-specific binding. The slides were then incubated overnight at 4°C with various combinations of two primary antibodies (Table S1), washed 3 times with PBS, and incubated with two secondary antibodies (Cy2-conjugated goat anti-mouse immunoglobulin G and Cy3-conjugated goat anti-rabbit immunoglobulin G) (both from Jackson Immunoresearch Laboratories) for 1 h at room temperature. The slides were mounted with FluorSave reagent (Merck Calbiochem), sealed and dried 1 h at room temperature. Images were captured with a Nikon Eclipse TE2000-E confocal laser-scanning microscope and analyzed with EZ-C1 3.70 imaging software (Nikon).

After fixation in 4% paraformaldehyde for 10 minutes and permeation using 0.2% Triton X-100 for 10 minutes, the IEC-6 cells were incubated with the HO-1 primary antibody (1∶200; Abcam) at 4°C overnight. The sections were incubated with CY3-conjugated goat-anti-rabbit immunoglobulin G (1∶200; Jackson, West Grove, PA, USA) at room temperature for 1 hour. All the sections were counterstained with DAPI nucleic acid stain (Invitrogen), and images were taken with an M1 Zeiss microscope under 400X magnification.