Fv 10 microscope

GFP expression in UAS-GFP/+; th-GAL4/+ organisms were used as a reporter for surviving dopaminergic neurons. Neuronal survival was quantified by dissecting out brains of the corresponding age and treatment. Brains were fixed with 4% formaldehyde in PBS, mounted individually in Citifluor (Ted Pella Inc. Redding, CA.) and images were taken at the National Laboratory of Advanced Microscopy using a confocal Olympus FV10 microscope with a 20X objective. Five brains of each genotype, age and condition were counted in a blinded manner. Confocal acquisition parameters were set and fixed using 10 days old control flies (UAS-GFP/+:Th-GAL4/+). the whole brain was sampled avoiding pixel saturation in the brightest section with optimal pinhole aperture and optimal section thickness, steps were 0.5X section thickness so the whole tissue was sampled twice, after data collection, maximum intensity projections (assembled with image J, https://imagej.nih.gov/ij/ taking in account section thickness and 2X oversampling) were used to make a single flat image and neurons were counted, if there was ambiguity about the number of neurons in a particular cluster in a particular maximally projected brain image, individual sections were analyzed. All other conditions and genotypes were acquired using these pre-set acquisition parameters.

CCM tissues were removed from the proband’s brain and fixed in formalin overnight at 4°C. The tissue was then embedded in paraffin wax and cut into 10-μm sections. Hematoxylin and eosin (H&E) staining was conducted using Harris-modified hematoxylin (Fisher Scientific) and alcoholic eosin (Fisher Scientific). The images were captured using Olympus FV-10 microscope.

DU145 cells were grown on 12-mm glass coverslips (Matsunami Glass Industry) in DMEM containing 10% FBS. The cells were cultured for 1 h in serum-free DMEM containing 2D5 peptide for 1 h and then stimulated with 100 ng/ml human recombinant EGF (PeproTech) for 20 min. The cells were fixed with cold methanol for 30 min and then LAMP-1 and EGFR were stained as described previously (15 (link)). Images were obtained by confocal microscopy under an FV-10 microscope (Olympus).