Axioskop2 mot plus wide field fluorescence microscope

For immunofluorescent labeling, a PAP pen was used to draw a hydrophobic barrier around the tissue ribbon. Sections were then incubated in 50 mM glycine in Tris buffered saline (TBS) for 5 minutes. TBS-glycine was aspirated off and a blocking solution containing 0.05% Tween, 0.1% bovine serum albumin (BSA) in TBS was applied for 20 minutes. Following blocking, primary antibodies in blocking solution were applied and coverslips were placed in a humidified chamber at 4°C overnight. The following day, tissue was washed in TBS and incubated in secondary antibody in blocking solution for 1 hour at room temperature protected from light. Tissue was then washed again in TBS and 5 µg/ml 4',6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI; cat #D1306, Invitrogen) in TBS was applied for 5 minutes followed by a final wash in TBS. Coverslips were mounted on glass slides in Vectashield fluorescent mounting medium (cat# H-1000, Vector Laboratories). See Tables 1 and 2 for a complete listing of primary and secondary antibodies and dilutions that have been tested in array tomography sections and optimized for immunofluorescent labeling of axons. All immunofluorescence was imaged using a Zeiss Axiovert 200 laser scanning confocal microscope with a 40 × 1.2 NA water immersion lens or a Zeiss Axioskop 2 MOT Plus wide-field fluorescence microscope with a 63 × 1.4 NA oil immersion lens.