Exosomes were processed for transmission electron microscopy (TEM) by negative staining with 2% uranyl acetate at the Electron Microscopy Unit (Facultad de Ciencias, Universidad de la República, Montevideo, Uruguay). Briefly, astrocyte exosome-enriched fractions were prepared as mentioned and resuspended in 30 µl PBS. 10 µl exosome suspension was added on top of carbon film-coated electron microscopy grids (Electron Microscopy Sciences) and incubated for 15 min, and excess liquid was removed with filter paper. Samples were incubated with 2% uranyl acetate for 5 min, with excess liquid removed as before, and let dry completely before imaging. Exosome preparations were examined using a Jeol JEM1010 TEM operated at 100 kV and equipped with 4,000 AM DVC and a HAMAMATSU C-4742-95 digital camera under the control software AMT ADVANTAGE. The exosome diameter was measured in electron micrographs using Fiji (ImageJ) software (NIH; RRID: SCR_002285).
C4742 95
Manufactured by Hamamatsu Photonics
Sourced in Japan, Germany, United States, Italy
The C4742-95 is a CCD camera system manufactured by Hamamatsu Photonics. It features a 1024 x 1024 pixel CCD image sensor with high quantum efficiency. The camera can capture images in a variety of resolutions and frame rates, making it suitable for diverse scientific and industrial applications.
Automatically generated - may contain errors
Lab products found in correlation
Axioplan 2, by Zeiss (7 mentions)
Openlab software, by PerkinElmer (7 mentions)
Hydromount solution, by National Diagnostics (5 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (5 mentions)
Eclipse te2000 u microscope, by Hamamatsu Photonics (4 mentions)
Dmi8 upright fluorescence microscope, by Leica (4 mentions)
Lsm 710, by Zeiss (4 mentions)
Axiocam mrm, by Zeiss (3 mentions)
Plan apochrome 63x na 1.4 oil immersion lens, by Zeiss (3 mentions)
Alexa fluor 488 conjugated donkey anti rabbit igg, by Jackson ImmunoResearch (3 mentions)
Rabbit anti ha, by Cell Signaling Technology (3 mentions)
Vectashield containing dapi, by Vector Laboratories (3 mentions)
Dmi6000b inverted microscope, by Leica (3 mentions)
Dmra2, by Leica (3 mentions)
Axioplan 2 microscope, by Hamamatsu Photonics (3 mentions)
Axioplan microscope, by Zeiss (3 mentions)
Mz16f, by Leica (2 mentions)
C11440, by Olympus (2 mentions)
Paraformaldehyde (pfa), by Thermo Fisher Scientific (2 mentions)
Sc 137021, by Santa Cruz Biotechnology (2 mentions)
83 protocols using c4742 95
1
Exosome Characterization by TEM
2
Fluorescence Analysis Using Microscopy
3
Visualizing GFP-nectin-1 Expression in K562 Cells
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K562 cells were fixed with paraformaldehyde at a final concentration of 3% about 48 h post electroporation with plasmid pCK495 [53 (link)]. Expression and localization of GFP-nectin-1 was assessed by observing GFP fluorescence using a Nikon Eclipse E600 equipped with a Hamamatsu C4742-95 camera.
4
Microscopic Imaging Protocol for Cellular Analysis
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Images were acquired using a microscope (Axioplan 2; Carl Zeiss, Inc.) with a Plan Apochrome 63x NA 1.4 oil immersion lens (Carl Zeiss, Inc.) and a digital camera (Hamamatsu Photonics, C4742-95). Images were acquired and processed using Openlab software (Perkin Elmer).
5
Immunofluorescence Imaging of PAEC Cells
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PAEC were grown on cover glass for three days after reaching 100% confluence, and fixed with 4% paraformaldehyde (Thermo Fisher Scientific) for 30 min, permeabilized with 100% cold methanol at −20 °C for 5 min. Cell then blocked with 1% BSA for 1 h, and later incubated with first antibody overnight at 4 °C then secondary antibody at room temperature for 1 h. Finally, cells were mounted on microscope slides using ProLong Glass Antifade Mountant, (Invitrogen, Carlsbad, CA, Cat# P36980). Immunofluorescent images were observed with a Nikon Eclipse TE2000-U microscope, with Hamamatsu digital camera C11440, and Olympus IX51 microscope with Hamamatsu digital camera C4742-95. The images were analyzed with ImagePro Plus 7.0 [29 ] or ImageJ software to evaluate the colocalization of fluorescent.
6
Microfluidic Neuronal Degeneration Assay
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Dissociated DRG neurons were plated in microfluidic chambers (150 μm barrier, XONA Microfluidics). Cell suspension was pipetted into each side of the upper channel of the microfluidic device. On DIV7, a difference of 100 μl of media between chambers was introduced and drugs were added to the compartment with the lower hydrostatic pressure. To calculate the % of viable neurons, 1 μg/ml propidium iodide (PI) (Thermo Fisher Scientific) was added to the media 15 min before drug addition. Phase contrast and fluorescence microscopy images were acquired on a DMi8 upright fluorescence microscope (Leica microsystems) coupled to a monochrome digital camera (Hamamatsu C4742-95). The objective used was HCXPL 20 X/0.40 CORR. For each experiment, the degeneration index was calculated from the average of two distal fields of neurites per condition, whereas the % of viable neurons remaining relative to the first time point was calculated from the average of three fields of cell bodies (staining positive for PI) per condition; the total number of experiments is indicated in the figure legends.
7
Immunofluorescence Imaging of DNA Repair Factors
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Previously published procedures were followed for IF and IF-FISH12 (link). IF for myc-tagged RPA32 or Ctc1 (mouse monoclonal, 9B11 or rabbit monoclonal, 71D10, Cell Signaling Technology), HA-tagged Stn1 (3724, Cell Signaling Technology), endogenous Polα (sc-137021, Santa Cruz), and 53BP1 (612522, BD Biosciences) was carried out using the cytoskeleton extraction protocol14 (link). Intensity measurements of RPA32-myc IF were performed in FIJI as follows: nuclei were identified using thresholding, segmented, and identified as regions of interest. The average image background was then subtracted from the image, and the total raw pixel intensity within each area of interest in the channel of interest was calculated. Rad51 (70-001, Bioacademia), and γH2AX (05636, Millipore) were detected in cells fixed in 3% PFA, and foci showing co-localization of Rad51 with γH2AX were quantified. IF imaging was performed on a Zeiss Axioplan II microscope equipped with a Hamamatsu C4742-95 camera using Volocity software or on a DeltaVision (Applied Precision) equipped with a cooled charge-coupled device camera (DV Elite CMOS Camera), a PlanApo 60× 1.42 NA objective or 100× 1.40 NA objective (Olympus America, Inc.), and SoftWoRx software.
8
Immunohistochemical Analysis of Cellular Markers
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Immunohistochemical (IHC) analyses were performed as previously described (Liang et al., 2012 (link); Chagani et al., 2017 (link); Li et al., 2017 (link)). Briefly, sections of 5 μm thickness were rehydrated and dissolved of paraffin with a graded xylene and ethanol series. Antigen retrieval was done with a citrate buffer pH 6.0. Blocking was done with 10% normal goat serum in PBS. Primary antibodies used were anti-CPD (Abcam, 1:1000) and anti-PCNA (Abcam, 1:6000) with incubation overnight at 4°C. Cy3 (Jackson Immuno Research, 1:400) was used as the secondary antibody. Nuclear staining was done using DAPI (0.2 ng/mL). Samples were then rinsed with PBST, dehydrated with graded ethanol and xylene washes, and mounted with distyene plasticizer xylene (DPX). Slides were allowed to dry overnight at room temperature before capturing images with Leica DMRA fluorescent microscope and Hamamatsu C4742-95 at 20x magnification using AxioVs40 version 4.8.2.0 software. Images were processed using Adobe Photoshop CC 2018 and Image J software version 1.50i.
9
Immunocytofluorescence Staining of Cell Cultures
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Immunocytofluorescence staining was performed at 24, 48 and 72 h of culture. Cells were washed in PBS then fixed and permeabilized in methanol at -20°C for 8 min and blocked for 1 h in 1% FFA BSA. Primary monoclonal antibodies (2.5 μg each) to desmoplakin (DSP, 0.05 mg/ml, Abcam) and cytoDEATH M30 (cCK18, 0.5 μg/ml, Roche) were prepared in PBS with 1% FFA BSA and incubated with the cells overnight at 4°C. Secondary antibodies (Alexa Fluor 488 or 555) were diluted in PBS with 1% FFA BSA and incubated with the cells for 1 h at room temperature. Samples were mounted in mounted-medium with DAPI for nuclear staining and photographed with a BX60 epifluorescence microscope (Olympus) equipped with a 40x oil objective (Olympus 1.00), an ultrahigh-vacuum mercury lamp and a Hamamatsu camera (C4742-95). Pictures were analyzed with VisionStage Orca software (v 1.6).
10
In Vivo Leukocyte Dynamics Imaging
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To label leukocytes, animals received a 100 μL bolus of Rhodamine 6G (5 mg/kg; Sigma, St. Louis, MO) 5 min before the time point of interest. Fluorescently labeled leukocytes were excited, and images were captured with a Vivid Set (XF104-2 filter, Omega Filters, Brattleboro, VT) using a high-light sensitive camera (C4742-95, Hamamatsu Photonics, Japan). 60 s of video was captured on a straight portion of the vessel at 10 frames per second. During playback, the vessels were segmented into 100 μm lengths, and leukocytes were counted and classified as “rolling” or “adhered” to the endothelium as previously described32 (link).
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