Wide field images were taken using an Olympus BX61WI fluorescence microscope (Olympus, Japan) on 20 µm cryo-sectioned tissue. Brightness and contrast were adjusted to balance the channels and background subtraction (ImageJ 1.51k, USA) was run on each image.
Bx61wi fluorescence microscope
Manufactured by Olympus
Sourced in Japan
The BX61WI fluorescence microscope is a high-performance research-grade microscope designed for fluorescence imaging applications. It features a motorized, infinity-corrected optical system and a versatile illumination system that supports a wide range of fluorescent probes and dyes. The microscope is suitable for a variety of applications in fields such as cell biology, neuroscience, and tissue engineering.
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Lab products found in correlation
Vt1200, by Leica (2 mentions)
Lsm 700 confocal microscope, by Zeiss (2 mentions)
Isopentane, by Merck Group (1 mentions)
Oct medium, by Bio-Optica (1 mentions)
Superfrost plus, by Thermo Fisher Scientific (1 mentions)
Sucrose, by Merck Group (1 mentions)
Imspector software, by Abberior (1 mentions)
0.95na water immersion objective, by Olympus (1 mentions)
Matlab, by MathWorks (1 mentions)
Axio imager z2, by Zeiss (1 mentions)
Zen blue, by Zeiss (1 mentions)
Volocity software, by Quorum Technologies (1 mentions)
Photomultiplier tube, by Hamamatsu Photonics (1 mentions)
Axioimager z2, by Olympus (1 mentions)
Donkey serum, by Merck Group (1 mentions)
Rabbit anti gaba antibody, by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Avantor (1 mentions)
Agarose, by Avantor (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
8 protocols using bx61wi fluorescence microscope
1
Wide-field Fluorescence Microscopy Analysis
2
Visualization of Dopaminergic Neurons
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Adult (5 females and 1 male, 12–16 weeks old) Vmat2-Cre;tdTomato mice were perfused (see below) using PBS followed by 4% formaldehyde (FA). Whole brain, spinal cord and dorsal root ganglia tissue were dissected out. Brains and two spinal cords were kept in 4% FA overnight at 4 °C, then mounted in 4% agarose (VWR) and cut in 60 µm thick sections using a vibratome (Leica VT1200). After the FA incubation, one spinal cord with intact dorsal root ganglia tissue was placed in 15% sucrose (Sigma-Aldrich, USA) in 1 × PBS for 24 h, followed by 24 h incubation in 30% sucrose in 1 × PBS for 24 h for cryoprotection. The tissue was thereafter embedded in optimal cutting temperature (OCT) medium (Bio-Optica, Milan, Italy) and snap-frozen on dry ice in − 80 °C isopentane (Sigma-Aldrich, Germany). The tissues were cut into 18 µm sections using a cryostat (Leica Cryocut 1800) and collected onto Superfrost Plus (Thermo Scientific) slides.
The slices were subsequently visualized using an Olympus BX61WI fluorescence microscope (Olympus, Japan), where red fluorescence (tdTomato expression) was imaged and edited using ImageJ (ImageJ, USA) and Adobe Photoshop (to create composites) software. Atlases38 ,39 were used to determine the anatomical position of the tissue.
The slices were subsequently visualized using an Olympus BX61WI fluorescence microscope (Olympus, Japan), where red fluorescence (tdTomato expression) was imaged and edited using ImageJ (ImageJ, USA) and Adobe Photoshop (to create composites) software. Atlases38 ,39 were used to determine the anatomical position of the tissue.
3
In Vivo Cell Migration Dynamics Imaged
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Mice were anesthetized and the mouse calvarium was surgically prepared as described previously (Pereira et al., 2009a (link)). Animals were immobilized on a custom-built stage. Deep-tissue images were collected using a BX61WI fluorescence microscope (Olympus) with a 20×, 0.95 NA water immersion objective (Olympus), and dedicated single-beam TriM laser-scanning microscope (LaVision Biotec), controlled by Imspector software (Abberior Instruments GmbH). The light source was a Chameleon Vision II Ti:Sapphire laser (Coherent) with pulse precompensation. For four-dimensional analysis of cell migration, stacks of 16–20 optical sections with 3-µm z spacing were acquired every 30 s for 30 min with the laser tuned to a wavelength of 895 nm. Each xy plane spanned 400 µm in each dimension. Videos were analyzed using Imaris, and cell tracks were automatically generated and then verified manually. Cell velocities and mean squared displacements were calculated using software programmed in MATLAB (The Math Works, Inc.)
4
Quantitative Fluorescence Imaging and Cell Counting
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Wide field images were taken using an Olympus BX61WI fluorescence microscope (Olympus, Japan) with a Volocity software (Quorum Technologies) or a Zeiss Axio Imager Z2 (Zeiss, Germany) with a colibri LED 7 and Zen blue software (Zeiss, Germany). Some images were taken using a Zeiss LSM700 confocal microscope (BioVis facility, Uppsala University). Brightness and contrast were adjusted in ImageJ, equally for the whole image and without obscuring any data. Images of brainstem were stitched using the Pairwise Stitching in ImageJ (ImageJ, RRID:SCR_003070) (Preibisch et al., 2009 (link)). Cell counts were done manually with ImageJ's Cell counter plugin (Schneider et al., 2012 (link)). For estimation of the total number of traced cells in the spinal cord, cells were identified and counted using an ImageJ macro based on signal intensity and size. A selection of automatically counted images was re‐counted manually, and the macro was found to identify 50–80% of cells.
Brain regions were identified based on the Allen Brain atlas and The Mouse Brain in Stereotaxic Coordinates (MBSC) (Franklin & Paxinos, 2008 ; Lein et al., 2007 (link)). Abbreviations followed the MBSC.
Brain regions were identified based on the Allen Brain atlas and The Mouse Brain in Stereotaxic Coordinates (MBSC) (Franklin & Paxinos, 2008 ; Lein et al., 2007 (link)). Abbreviations followed the MBSC.
5
Live Imaging of Popliteal Lymph Nodes
6
Optimized Immunohistochemistry and RNAscope Imaging
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Images of immunohistochemistry treated sections were acquired using a wide-field Olympus BX61WI fluorescence microscope (Olympus) with a 10× objective, for which the brightness and contrast were optimized for each channel during image acquisition and quantification. The RNAscope treated sections were acquired with wide-field 20× magnification with an Olympus BX61WI fluorescence microscope (Olympus) or an Axio Imager.Z2 (ZEISS), where each channel was set to be automatically optimized for each image, but had to be further optimized during image analysis. Here, the optimal intensity and contrast was set for one image (reference image) and the settings of the other images were set to match the reference image. The images were manually quantified using the Fiji (ImageJ 1.52f) Cell Counter plug-in.
7
GABAergic neuron identification in Vmat2-Cre mice
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One 16-week old male and two 14-week old female Vmat2-Cre;tdTomato mice were perfused with PBS followed by 4% FA. The whole brain was dissected out and kept in 4% FA overnight at 4℃. Before slicing, the tissues were mounted in 4% agarose (VWR) and cut into 60 µm thick sections using a vibratome (Leica VT1200). Immunostaining with rabbit anti-GABA antibody (Sigma-Aldrich) 1:750 in blocking solution (5% Donkey Serum (Sigma-Aldrich), 3% BSA (Sigma-Aldrich) in TBS) was performed (10 sections per animal). Cells were stained with DAPI (VWR) and Donkey anti-rabbit 647 antibody (Invitrogen) for visualization. The stained sections were mounted and imaged using an Olympus BX61WI fluorescence microscope (Olympus, Japan) with 10 × magnification. Some images were merged to increase the focus using ImageJ (ImageJ, USA) software. Only tdTomato cells with clear DAPI expression were counted. The result is presented as percentage ± SEM of Vmat2-Cre;tdTomato cells expressing GABA.
8
RNAscope Quantification of Vmat2-Cre Neurons
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Images of RNAscope treated Vmat2-Cre.HTB SB1F sections were acquired with wide field 20 × magnification using an Olympus BX61WI fluorescence microscope (Olympus, Japan). The channel for each probe was optimized for brightness and contrast during image acquisition and quantification. The RNAscope images were manually quantified using the Fiji (ImageJ 1.52f) Cell Counter plugin52 (link),53 (link). All Vmat2-Cre.HTB cells with DAPI overlap were considered cells and one read of the targeted probe could be visualized as one dot. A Vmat2-Cre.HTB cell was considered to be expressing the targeted gene if the overlapping # dots ≥ 3. The result is presented as percentage ± SEM of Vmat2-Cre.HTB cells expressing Vmat2, Vglut1, Vglut2, Viaat, Rorb and Scnn1a.
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