Adult male C57 mice (P102) were anesthetized by isoflurane and decapitated. The brains were rapidly removed and frozen in medium (Tissue-Tek) on dry ice. Coronal brain slices (25μm) containing A1 were cut with a cryostat (Leica CM 1900), adhered to SuperFrost Plus slides (VWR), and immediately refrozen on dry ice. In situ hybrization was performed using the protocol provided by the RNAscope Multiplex Assay manual (Advanced Cell Diagnostics). The following fluorophore-conjugated RNAscope probes were used: Mm-Lynx1-C1 (Cat No. 449071), Mm-Htr3a-C2 (Cat No. 411141), Mm-VIP-C2 (Cat No. 415961), and Mm-Chrna4-C3 (Cat No. 429871). Confocal images were acquired with a Zeiss LSM 710 confocal using a 63x, 1.4NA oil immersion PlanAPO objective (1.1x zoom).
Planapo
Manufactured by Zeiss
Sourced in Germany
The PlanApo is a high-quality objective lens designed for advanced microscopy applications. It delivers exceptional optical performance, including a flat image field, high numerical aperture, and excellent chromatic correction. The PlanApo is suitable for a wide range of research and industrial applications that require precise and detailed imaging.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 710, by Zeiss (4 mentions)
Lsm 510 meta confocal microscope, by Zeiss (4 mentions)
Axiovert 200m, by Zeiss (4 mentions)
Lsm 510, by Zeiss (4 mentions)
Rnascope multiplex assay manual, by Advanced Cell Diagnostics (2 mentions)
Superfrost plus slides, by Avantor (2 mentions)
Cm1900, by Leica camera (2 mentions)
Zen 2012, by Zeiss (2 mentions)
Observer 1.1 microscope, by Zeiss (2 mentions)
Emccd image mx2 camera, by Hamamatsu Photonics (2 mentions)
N sim microscope, by Nikon (2 mentions)
Prolong gold mounting medium, by Thermo Fisher Scientific (2 mentions)
Axio observer z1 microscope, by Zeiss (2 mentions)
Efficient navigation zen , by Zeiss (2 mentions)
Lsm 510 meta, by Zeiss (2 mentions)
Lsm 880, by Zeiss (2 mentions)
Lsm 780, by Zeiss (2 mentions)
Lsm 700, by Zeiss (2 mentions)
Matlab r2020a, by MathWorks (1 mentions)
Dv401 bv, by WITec (1 mentions)
28 protocols using planapo
1
Multiplexed in situ hybridization of mouse A1 cortex
2
Quantitative Confocal Imaging of Olfactory Bulb
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Mounted sections were imaged on a LSM 700 Zeiss confocal system using a 20x dry objective (PlanApo, NA = 0.8) using 405, 488, 555 and 633 laser lines. Confocal z‐stack images were acquired. Images were processed with Zen 2012 (Zeiss, Oberkochen, Germany) and ImageJ/FIJI software. Quantifications were performed on 3 individual 4.0 × 10−4 mm3 volumes within the olfactory bulb in 3 animals. These volumes were chosen in the central areas of the olfactory bulb that contained a similar density of Hoechst‐positive nuclei across all imaged brain slices. Average numbers of three individual volumes per fish were quantified. Student's t‐tests were carried out on the averages of measurements in at least 3 individual animals (n = 3 or n = 4) were used to determine P‐values.
3
Raman Spectroscopy Analysis of Polymeric Microspheres
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The PS microspheres on a SiN film or slide glass were observed under a confocal laser Raman microscope using a 100× objective lens (Plan apo, Carl Zeiss, Oberkochen, Germany) and a 532-nm Nd-YAG laser (alpha300R, WITech, Ulm, Germany). Spectra were acquired with a Peltier-cooled charge-coupled device detector (DV401-BV, Andor, UK) with 600 gratings/mm (UHTS 300VIS, WITec, Germany). WITec suite (version 7.0, WITec, Germany) was used for data acquisition. For 2D Raman spectra, the laser intensity was 1.5–3 mW and the number of pixels in the XY axis was from 120 × 120 to 300 × 300. The pixel step width was 100 nm, and the measurement time for each pixel was set to 0.05 s. XZ axis scans were performed from 120 × 60 pixels, the step width set to X axis of 100 nm and Z axis of 200 nm. Raman spectral data were calculated using MATLAB R2020a (Math Works Inc., Natick, MA, USA) and plotted using Origin 2021J (Origin-Lab Co., Northampton, MA, USA).
4
Sensitive Microscopy of HeLa Cells
5
Immunofluorescence Microscopy of Cellular Structures
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Immunofluorescence (IFA) microscopy was performed with formaldehyde fixed/detergent permeablized cells as described in [55 (link)]. Cells were also stained with DAPI (0.5 μg ml-1) to reveal nuclei and kinetoplasts. Serial image stacks (0.2 micron Z-increment) were collected with capture times from 100–500 msec (100x PlanApo, oil immersion, 1.46 na) on a motorized Zeiss Axioimager M2 stand equipped with a rear-mounted excitation filter wheel, a triple pass (DAPI/FITC/Texas Red) emission cube, differential interference contrast (DIC) optics, and an Orca ER CCD camera (Hamamatsu, Bridgewater, NJ). Images were collected with Volocity 6.1 Acquisition Module (Improvision Inc., Lexington, MA) and individual channel stacks were deconvolved by a constrained iterative algorithm, pseudocolored, and merged using Volocity 6.1 Restoration Module. Unless otherwise stated all images presented are summed stack projections of merged channels. The xyz pixel precision of this arrangement has been validated in [18 (link)] (see S1 Fig therein).
6
Immunofluorescence Staining of Focal Adhesions
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Cells were fixed on 1.5mm glass coverslips with PHEMO buffer (68 mM, PIPES, 25 mM HEPES, 15 mM EGTA, 3 mM MgCl2, 10% DMSO) plus 3.7% formaldehyde, 0.05% glutaraldehyde and 0.5% Triton X-100 for 10 min at room temperature. After three 5 min washes with PBS, the coverslips were blocked with 10% normal goat serum for 1 h at room temperature. Primary antibodies were diluted in 5% normal goat serum and added to the coverslips overnight at 4 °C. After 3 PBS washes, the coverslips were incubated for 1 h at room temperature with a fluorophore conjugated secondary antibody diluted in 5% normal goat serum. After 3 PBS washes, the coverslips were incubated with 350 nM DAPI stain diluted in 1X PBS for 10 min at room temperature. The coverslips were mounted on glass slides using Prolong gold mounting medium (Invitrogen). The slides were imaged using Zeiss LSM 510 META confocal microscope using a 100X oil Plan-Apo objective (NA = 1.46). The images were processed using ZEN 2009 software. A Nikon N-SIM microscope housed within the Emory Integrated Cellular Imaging Core was utilized to acquire super-resolution images of vimentin directly entering FAs. Images were acquired using a 100× 1.49 NA oil objective. All images within an experiment were taken under identical settings. Intensity levels were adjusted equally on all samples within an experiment.
7
Immunofluorescence Staining of Focal Adhesions
8
Confocal Imaging of Cellular Scaffolds
9
Quantifying Midbrain Dopaminergic Neurons
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Dopaminergic TH+ neurons were observed using an inverted fluorescence microscope (Axiovert 200M, Zeiss) under a 10× objective (PlanApo, NA = 0.45). The diameter of every well was scanned in two perpendicular directions (i.e. top to bottom and left to right) and total TH+ neurons were counted for every well.
10
CPC Phase Separation and Condensate Dynamics
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CPC phase separation was induced by incubating 1 μM CPC in a buffer containing 150 mM NaCl and 20 mM Hepes (pH = 7.2), or a buffer composed of 500 mM NaCl and 20 mM Hepes (pH = 7.2) supplemented with 7% PEG3350 to serve as a crowding agent (Fig. 1 C). For consistency, in all other experiments, CPC phase separation was induced by mixing with a buffer composed of BRB40 (40 mM Pipes, 0.5 mM MgCl2, 0.5 mM EGTA, pH 6.8), 1 mM GTP, 1 mM DTT, and 7% PEG3350. For time-lapse imaging of CPC droplet fusion, CPC phase separation was induced with a BRB40 mix containing 7% PEG3350, the mixture was transferred to microscopic chambers, and droplet fusion was immediately imaged under ×63 objective (Plan Apo) DIC on a ZEISS AxioObserver Z1 microscope.
To compare the phase separation capacity of CEN-BorealinLLPS and CEN-Borealinwt, phase separation of each protein was induced by diluting CEN into buffer containing 150 mM NaCl. A sample from this mixture was immediately transferred into a coverslip flow chamber made of small channels separated by paraffin wax. DIC images of condensates were acquired after a 15 min incubation of this chamber at room temperature, which allowed condensates to settle to a common Z plane as well as to relax and fuse.
To compare the phase separation capacity of CEN-BorealinLLPS and CEN-Borealinwt, phase separation of each protein was induced by diluting CEN into buffer containing 150 mM NaCl. A sample from this mixture was immediately transferred into a coverslip flow chamber made of small channels separated by paraffin wax. DIC images of condensates were acquired after a 15 min incubation of this chamber at room temperature, which allowed condensates to settle to a common Z plane as well as to relax and fuse.
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