Brain slices were prepared as described above. Interneurons expressing eGFP were identified visually using epifluorescent optics on a Nikon E600FN upright microscope (Melville, New York, USA). Patch electrodes (4–6MΩ) were filled with internal solution containing 0.4% biocytin. Cells were patched for 15–25 minutes to allow for adequate filling of the processes. After filling, slices were immediately placed in 4% PFA for 24–72 hours and stored in PBS until processing. Slices were washed in PBS, incubated in 10% methanol and 3.5% H2O2 in PBS for 10 minutes, washed in PBS, and incubated in TRITC conjugated steptavidin (Jackson Immunoresearch, West Grove, PA) in 0.3% PBST for two hours. After washes with PBS, slices were mounted onto charged microscope slides and coverslipped with Prolong Antifade Gold (Invitrogen). Neurolucida software (MBF Bioscience, Williston, VT, USA) was used to trace biocytin-labeled interneurons. As axons were not reliably labeled, only soma and dendrite characteristics were measured. Variables of interest included soma size, dendrite length and volume, and number of dendrites, branch points, and branches.
E600fn upright microscope
Manufactured by Nikon
Sourced in United States
The E600FN upright microscope is a laboratory equipment designed for high-quality optical imaging and analysis. It features a stable and durable construction, as well as advanced optical components for clear and detailed observation.
Automatically generated - may contain errors
Lab products found in correlation
Prolong gold antifade, by Thermo Fisher Scientific (1 mentions)
Neurolucida software, by MBF Biosciences (1 mentions)
Orca 2 er ccd, by Hamamatsu Photonics (1 mentions)
Multiphoton microscope, by Nikon (1 mentions)
Lasersharp software, by Bio-Rad (1 mentions)
Fluo 4, by Thermo Fisher Scientific (1 mentions)
Ti sapphire infrared laser, by Spectra-Physics (1 mentions)
3 protocols using e600fn upright microscope
1
Interneuron Morphological Analysis
2
Spinning Disk Confocal Microscopy for Live Cell Imaging
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Live cell imaging was performed with a spinning disk confocal microscope, which has been shown to create very low photobleaching [27 (link)]. We used the Yokogawa CSU-10 confocal scan head (www.yokogawa.com ), attached to a Nikon E600FN upright microscope equipped with a PlanApo 100×/1.45NA DIC objective lens (www.nikonusa.com ). An Argon-Krypton ion laser with ~10 mW power at 488 nm excitation illuminated the scanning unit through an optical fiber (www.mellesgriot.com ). The effective laser power which reached the cells was ~0.2 mW. Images were captured with a cooled back-thinned ORCA-II-ER CCD (charge coupled device) camera (www.hamamatsu.com ) controlled by MetaMorph 6.0 (www.moleculardevices.com ) running on Windows XP.
3
Two-Photon Ca2+ Imaging of Neuronal Dynamics
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Two photon Ca 2+ imaging was performed with a Bio-Rad multiphoton microscope based on a 1024 scan head installed on a Nikon E600FN upright microscope with a mounted recording chamber and a Nikon 60x NA 1.0 water-immersion objective. A Millennia V pump laser coupled to a mode-locked Ti:sapphire infra-red laser (Tsunami, Spectra Physics) was used for fluorescence excitation, tuned to 790 nm. Imaging parameters and controls were as described previously (Tonini et al., 2013) (link). In short to observe Ca 2+ transients, 20 µM Fluo-4 (Molecular Probes, Eugene, Oregon) was dissolved in the intracellular recording solution. Upon achieving the whole-cell configuration, the dye was allowed to equilibrate for 10-15 min in the proximal dendrites of the imaged neuron. Line scans images (Lasersharp software; Bio-Rad, UK) triggered by electrical stimuli delivered at the soma were collected from proximal apical processes (<50 µm from the soma) at 6 ms intervals.
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