Immunocytochemistry and microscopy.
Retina dissections, radial cryosections (~6 μm), and immunohistochemistry procedures were carried out as described elsewhere [19]. Brains from 3- to 5-mo-old C57Bl/6 mice were fixed overnight in 2% paraformaldehyde, infused with 30% sucrose, and processed for cryosectioning. Primary brain neurons and glial cells were prepared from the cerebral cortex. This was macerated in the Hank's Balanced Salt solution and triturated. Primary cells were cultured in DMEM (GIBCO, San Diego, California, United States) and collagen-coated 35-mm glass bottom culture dishes (MatTek Corporation, Ashland, Maine) at 5% CO2/37 °C for ~2 d and processed for immunocytochemistry. Primary antibodies were used at concentrations ~2.5–10 μg/ml. Alexa 488- and Alexa 594-conjugated secondary antibodies (2.5 μg/ml) (Molecular Probes) were used for visualization of proteins. Crossover of fluorescent probes, background, and autofluorescence were found to be negligible. Visualization of specimens and localization of proteins were carried out by wide-field epifluorescence microscopy on Nikon (Tokyo, Japan) E600 upright and TE2000U inverted research microscopes equipped with similar Apochromat objectives. Images with the Nikon E600 were acquired with a SPOT-RT digital camera coupled to the microscope and driven by SPOT Imaging v4.0 software (Diagnostic Instruments, Sterling Heights, Michigan, United States). All images were captured at nonsaturating integration levels, 12-bit mono black/white, and then pseudo-colored. Protein colocalization analysis was carried out on the Nikon TE2000U microscope equipped with appropriate excitation and emission filter wheels, cube filters, 100 W mercury light source, Nomarski/DIC, and Plan Apochromat optics (100×, 60×, and 40× oil objectives with NA of 1.4, 40 ×LWD, and 20 ×LWD objectives and encoded motorized Z-Stage (Prior Scientific, Rockland, Maryland, United States). Images with the inverted Nikon TE2000U microscope were obtained using a CCD camera (CoolSNAP HQ; Roper Scientific, Trenton, New Jersey, United States). Images acquired under identical acquisition parameters were analyzed using Metamorph Software v6.2 (Universal Imaging, Glendale, Wisconsin, United States). Whenever applicable, serial optical Z-stacks (20–30 focal planes at 100-nm intervals) were captured and computationally processed by 3-D blind deconvolution methods with the same software.