Canadian Health&Care; Mall

Sections were washed in PBS (3X for 5 min) and then blocked in PBS containing 1% BSA for 15 min. Primary antibodies were applied in a moist chamber for 90 min at room temperature or overnight at 4°C. Sections were washed in high-salt PBS (2.7% NaCl) twice for 5 min and once in normal PBS. Secondary antibody was then applied for 1 h at room temperature followed by washes as described above. Slides were mounted in Vectashield medium (Vector Laboratories, Inc., Burlingame, CA). Some sections were double-stained by subsequent incubation with another primary antibody raised in a different species, followed by an appropriate secondary antibody. Digital images were acquired using a Nikon Eclipse 800 epifluorescence microscope (Nikon Instruments, Inc., Melville, NY) using a Hamamatsu Orca 100 CCD camera (Hamamatsu, Bridgewater, NJ), analyzed using IPLab scientific image processing software (Scanalytics, Inc., Fairfax, VA), and imported into Adobe Photoshop image editing software (Adobe Systems Inc., San Jose, CA). Some images were taken using a Radiance 2000 confocal microscopy system (Bio-Rad Laboratories, Hercules, CA) using LaserSharp 2000 version 4.1 software and were imported into Adobe Photoshop software as TIFF files.