AAV5-EF1a-DIO-ChR2 (H134)-eYFP virus was purchased from University of North Carolina, Vector Core Facilities, and delivered to the VTA by stereotaxic surgery as previously described (Bimpisidis et al., 2019 (link), 2020 (link); Figure 1A ) in order to visualize VTA NEX-Cre positive neurons and their projections. Briefly, NEXCre+/wt mice (>8 weeks old; >20 g) were deeply anesthetized with isoflurane and received 300 nl of virus in the right VTA (AP: −3.45 mm, L: −0.2 mm, V:-4.4 mm according to Franklin and Paxinos, 2008 ) at 100 nl min−1 flow rate. Four weeks after injection the mice were transcardially perfused, their brains were collected and cut in a vibratome at 30um-thick sections. The sections were mounted, coverslipped and imaged using a Leica epifluorescent microscope.
Epifluorescent microscope
Manufactured by Leica camera
Sourced in Germany, Japan
The Epifluorescent microscope is a type of microscope that uses fluorescence to illuminate and observe samples. It is designed to detect and analyze the distribution and intensity of fluorescent markers within a specimen. The core function of the Epifluorescent microscope is to provide high-contrast, high-resolution imaging of fluorescently labeled samples.
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6 protocols using epifluorescent microscope
1
Optogenetic Targeting of VTA NEX-Cre Neurons
2
Quantitative Biofilm Enumeration Protocol
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All medium was removed and biofilms washed twice using diluted BHI. Biofilms were resuspended in 1 ml Hank’s balanced salt solution (HBSS) as described [13] (link). Briefly, bacterial suspensions (n = 3) underwent 10-fold dilutions in HBSS and 5×20 µl of each dilution spot plated onto CBA plates and incubated at 37°C/5% CO2. For total cell counts (n = 3) bacterial suspensions were diluted 10-fold, stained with Syto 9 (Life Technologies, U.S.A.) according to manufacturer instructions, and individual cells counted using a haemocytometer (Marienfeld-Superior, Germany) and a Leica epifluorescent microscope using a 100x oil immersion lens.
3
Immunophenotyping of Cultured Cells
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Cells were seeded on a fibronectin-coated slide in a 48-well plate. After methanol-ethanol (v/v) fixation, cellular membranes were permeabilized with the permeabilization reagent 0.2% Triton X-100 in PBS. Nonspecific binding sites were blocked with 10% FBS. Cells were then incubated with primary antibodies against CD31, CD146, α-SMA, NG2 and PDGFRβ. Cell nuclei were counterstained with DAPI. References, manufacturers and concentrations of primary antibodies are listed in the Supplementary Table S2 . All image acquisitions were done with the NIS-Elements software (Nikon, Tokyo, Japan, V3.22.10) coupled with a Leica epifluorescent microscope.
4
Fixation and Imaging of Electroporated Brains
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Electroporated brains were dissected at the indicated embryonic ages, and successful electroporations were identified under an epifluorescence microscope. Brains with fluorescent labeling in the somatosensory cortex were fixed in a 4% formalin/PBS solution overnight at 4°C and cryoprotected in a 30% sucrose/PBS solution. Brains were frozen in optimal cutting temperature compound before 14-μm-thick coronal sections were obtained with a cryostat and placed on slides. Brain tissue was counterstained with DAPI before coverslipped with Fluoromount G mounting media. Most images were obtained with a Leica epifluorescent microscope on a 10× objective and captured with LAS X software. Images of three consecutive brain slices per brain were acquired.
To acquire high-magnification images, we used an Olympus Fluoview 3000 confocal laser scanning system on a 20× objective. Images were imported into Fiji for subsequent file conversion to TIFF format (RapID) or manual quantification (Cell Counter). For shCul5 results, raw images from a previously published study (Simó et al., 2010 (link)) were used.
To acquire high-magnification images, we used an Olympus Fluoview 3000 confocal laser scanning system on a 20× objective. Images were imported into Fiji for subsequent file conversion to TIFF format (RapID) or manual quantification (Cell Counter). For shCul5 results, raw images from a previously published study (Simó et al., 2010 (link)) were used.
5
Neuroanatomical Mapping of ChR2 Expression
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After behavioural testing, mice were deeply anesthetized with pentobarbital (Fatal Plus, 390 mg/ml) and transcardially perfused with 1x phosphate buffered saline (PBS) followed by 4% paraformaldehyde. Brains were removed, placed in vials containing 4% paraformaldehyde overnight at 4 °C, and were then transferred to a 20% (1 day) and then 30% sucrose solution until brains had sunk and ready for slicing. A sliding microtome (Leica Biosystems) was used to cut 30 µm coronal sections, which were subsequently slide mounted and imaged with an epifluorescent microscope (Leica) to identify location of ChR2 virus and optical fiber placement. Only data from mice with correct virus and fiber placement were used for analysis.
6
Quantifying Heterotrophic Nanoflagellates
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Samples for the measurements of heterotrophic nanoflagellate (HNF) abundance were fixed immediately after sampling with 1% glutaraldehyde (final concentration). Primulin-stained HNF collected on 0.8-µm polycarbonate black filters (25 mm diameter) were counted under UV excitation under a LEICA epifluorescent microscope as described by Caron (1983) . A total of 200-400 nanoflagellates from each slide were counted along several transects (SD < 10%). All solutions were filter-sterilized, and a blank was routinely examined to control for contamination of equipment and reagents.
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