Avidin d

Mice were perfused with Tyrode’s solution followed by 4% paraformaldehyde in PBS. The spinal column was removed, post-fixed, decalcified, cryoprotected, embedded and frozen in OCT (Cellpath, Newton, UK). 12 μm sections were blocked with Avidin D and biotin (Vector Labs, Burlingame, CA), and stained with the following primary and secondary antibodies: goat anti-MBP (1:300, Millipore), Alexa Fluor 488-conjugated rabbit anti-goat IgG (1:300, Life Technologies), biotinylated rat anti-CD45 (1:100, eBioscience), and Alexa Fluor 647-conjugated streptavidin (1:300, Life Technologies). Nuclei were labeled with DAPI (Life Technologies). Images were acquired on a Nikon A1 confocal microscope (Nikon, Tokyo, Japan).

Before intracellular staining, samples were incubated with Avidin-D (10ug/ml, Vector laboratories) in staining buffer for 15 minutes on ice to saturate all unbound surface biotin. Subsequently, cells were prepared for intracellular staining using an intracellular staining kit (eBioscience). Cells were stained with streptavidin-Alexa647 to visualize the intracellular biotin-MHCII. Ab to EEA1, Lamp1, and RAB5 or RAB7 followed by the appropriate secondary stain were included for colocalization studies at this point (table III). At the end of the staining, cells were fixed in 1% formaldehyde in PBS for 20 minutes, washed, and resuspended in PBS for acquisition.

Ten-micrometer cryostat coronal sections of vocal folds were prepared and air dried. Double immunohistochemical staining was performed to detect HA and collagen type III in normal and scarred vocal folds, with nuclear counterstaining by TOTO-3 (Molecular probes, Eugene, OR). Sections were fixed for 1 minute at room temperature in 4% paraformaldehyde (PFA), and washed three times in phosphate-buffered saline (PBS). Sections were then blocked with 5% normal goat serum in 0.1% Triton-X in PBS for 1 hour, then incubated overnight at 4°C with mouse monoclonal anti-collagen type III antibody (1:4,000; Sigma-Aldrich, St. Louis, MO) and biotinylated HA binding protein (2.5 μg/mL; Seikagaku Co., Tokyo, Japan) with 1% normal goat serum in 0.1% Triton-X. Next day, the sections were washed in PBS and incubated for 1 hour with Cy3-conjugated anti mouse IgG (1:400; Amersham Biosciences, Piscataway, NJ), Avidin-D (1:1000, Vector Labs, Burlingame, CA), and TOTO-3 (200 nM). Finally, samples were washed three times in PBS, mounted in Vectashield and cover-slipped (Vector Labs) for observation under a laser-scanning confocal microscope (Bio-Rad H600, Hercules, CA). Rat skin was used as a positive control for each staining process. Omission of the primary antibody served as a negative control.