The type of measurement that shows the maximum difference between the relatively healthy left kidney and the diseased right kidney over the progression of the UUO was the SHG channel data acquisition, when analyzed without background subtraction. The optical filters used in this channel, combination of BG39 (blue (2) in Figure S1 ) and UG11 (black (1) in Figure S1 ), is meant to primarily separate SHG signal (green (4) in Figure S1 ). In these measurements the samples were excited with 710 nm excitation laser line using a two-photon excitation scheme. The optical filter can pass short wavelength fluorescence from 360 nm to 410 nm (light blue shaded area in between UG11 (black) and BG39 (blue) in Figure S1 ). To identify the origin of this short wavelength fluorescence signal the average fluorescence spectra of the tissue samples were measured using an Olympus FV1000 microscope equipped with a grating-based spectrograph (Andor SR303i) with a 512-channel ultrafast EMCCD (Andor iXon Ultra). The optical restrictions associated with the Olympus FV1000 microscope set up prohibit measurements below 360 nm.
Fv1000 microscope
Manufactured by Olympus
Sourced in Japan, United States, United Kingdom
The FV1000 is a high-performance laser scanning confocal microscope developed by Olympus. It offers advanced optical and imaging capabilities for scientific research and investigation. The FV1000 provides precise control over the illumination and detection of fluorescent samples, enabling detailed visualization and analysis of cellular and subcellular structures.
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Lab products found in correlation
Prolong gold antifade reagent, by Thermo Fisher Scientific (6 mentions)
Axio observer z1, by Zeiss (6 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (4 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (4 mentions)
Lsm 700, by Zeiss (4 mentions)
Fluoview 1, by Olympus (3 mentions)
Fluoview version 2, by Olympus (3 mentions)
Leica dmi6000b microscope, by Leica Microsystems (2 mentions)
Fv1000 confocal microscope, by Olympus (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Sr303i, by Oxford Instruments (2 mentions)
Ixon ultra, by Oxford Instruments (2 mentions)
Gfp 1020, by Aves Labs (2 mentions)
Uplansapo, by Olympus (2 mentions)
Uv 3600 spectrophotometer, by Shimadzu (2 mentions)
Ab152, by Merck Group (2 mentions)
Imagej software, by Olympus (2 mentions)
Slowfade gold antifade reagent with dapi, by Thermo Fisher Scientific (2 mentions)
Prolong diamond antifade mountant, by Thermo Fisher Scientific (2 mentions)
Fugene hd, by Promega (2 mentions)
233 protocols using fv1000 microscope
1
Kidney Fibrosis Progression Monitoring
2
Fluorescent Imaging of Phosphorylated RNA Polymerase II
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All steps except for the imaging were carried out at 4°C. For phosphorylated RNA polymerase II detection, fixed embryos were washed in PBTw (PBS with 0.1% Tween-20) for 3×20 min, followed by permeabilization in PBTr (PBS with 0.1% Triton X-100) for 3×30 min and by blocking in blocking solution (PBS with 1% DMSO, 0.1% Triton X-100 and 5% skimmed milk). Blocked embryos were incubated overnight with anti-RNA polymerase II CTD pSer5 (1:1000, Abcam Cat# ab5131), washed in PBTw, reblocked in blocking solution and incubated in the secondary antibody (Alexa Fluor 594 goat anti-rabbit IgG(H+L) (1:300, Molecular Probes Cat# a11012) before imaging. DAPI (Molecular Probes, D1306) and 36/E-Cadherin antibody (BD Transduction Laboratories, 610182) were used for whole-mount nuclear and E-cadherin staining, respectively. Yolk cell membrane was marked using rhodamine-labeled wheat germ agglutinin (WGA) (Vector Laboratories, Cat# RL-1022). Whole-mount fluorescence images were acquired using Olympus FV1000 microscope. Section fluorescence images were acquired using Olympus BX51W1 or Olympus FV1000 microscope after processing for 10 µm paraffin-embedded section or 12 µm cryosection followed by mounting in ProLong Gold antifade reagent with DAPI (Molecular Probes Cat# P36931). Mitotic division angles were measured using Fiji software and data were plotted as rose diagrams using GeoRose software.
3
Histological and Immunological Assessment of Liver Fibrosis
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Liver tissues were immediately formalin fixed, paraffin embedded, and 10 μm thick slices were stained with Masson's Trichrome for a histological assessment. Immunohistochemstry and Immunofluorescence staining with α-SMA(at a 1:100 dilution, Abcam, ab21027) and anti-MAT2A (at a 1:50 dilution, NOVUS, NBP1–92100) were also performed to the species. Immunofluorescence staining was examined using the confocal laser scanning microscopy (Leica Microsystems Heidelberg GmbH, Germany). Stained sections were examined using an Olympus FV1000 microscope (Olympus Corporation, Tokyo, Japan). Image-Pro® plus 4.5 image analysis software (Media Cybernetics, USA) was used to quantify the degree of fibrosis. All of these experiments were completed by KCI BioTech (Suzhou, China).
4
Imaging Worm Fluorescence Microscopy
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Worms were anesthetized in 20 mM sodium azide in M9 and immobilized on 2% agarose pads. Fluorescent images were taken with Zeiss Observer D1 microscope 40 × 0.6 lens, excitation 480 nm LED light source, Confocal images were takes with Olympus FV1000 microscope (Olympus) 60 × 1.4 oil immersion lens. Excititation 440 nm. The images were processed with ImageJ v 1.51n.
5
Microscopic Imaging Techniques
6
Histological Analysis of HeLa Xenograft Tumors
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For histological analysis, the HeLa cell bearing nude mice were sacrificed on 21 days after injection of tested samples, the major organs and tumors were collected and fixed in 4% paraformaldehyde overnight. The paraffin embedded specimens were cut at 5 μm-thick sections, de-waxed and rehydrated with xylene, a graded series of ethanol, and deionized water successively.40 (link) Finally, the fixed sections were stained with H&E to assess histological alterations by OLYMPUS FV 1000 microscope (OLYMPUS, Japan).
7
Optogenetic Activation of Motor Neurons
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Single-cell photostimulation of MN2 after St.27 (swimming larva) was performed with Channelrhodopsin-2 (hChR2; reference (Zhang et al., 2007 (link)) transduced larva. pSP-Neurog-hChR2(E123T/T159C):mCherry were electroporated to express ChR2 at MN2L and MN2R. Laser irradiation was performed by a CLSM Olympus fv1000 microscope. After St.24, pSP-Neurog-hChR2(E123T/T159C):mCherry was expressed exclusively in the prRNs, MN2s, MN1s, and MGIN1s (depending on the larva due to mosaic expression). Therefore, to avoid photostimulating neurons other than MN2s, the region of interest (ROI) for laser irradiation was restricted to the targeted neuron. Stimulation was performed by irradiation with a 488 nm laser for 0.3 s (laser power density 15 μW/cm2).
8
Immunohistochemistry Protocol for Mouse Tissues
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For immunohistochemistry, Bl/6 (n = 32) and D2 (n = 33) eyes and olfactory epithelium (Bl/6 n = 4) were cryo-protected in 30% sucrose overnight at 4°C, embedded in Tissue Freezing Medium (Triangle Biomedical, Durham, NC) and then sectioned on a cryostat (Fisher, Pittsburgh, PA). We collected 10 μm-thick sections on 12 slides, with each slide containing representative sections from the entire eye. The sections were rinsed with PBS and incubated at room temperature in normal donkey serum at 1:20 in 0.1 M phosphate buffer with 0.5% bovine serum albumin and 0.1% Triton X 100 (PBT) for 2 hours, followed by an overnight incubation at 4°C in primary antibody in PBT (Table 1 ). The sections were rinsed with PBS and incubated with a secondary antibody at a 1:200 dilution (Life Technologies, Grand Island, NY) for 2 hours at room temperature, rinsed with PBS and mounted in Vectashield Mounting medium with DAPI (Vector Laboratories, Burlingame, CA). Imaging was performed on a Nikon Eclipse epifluorescence microscope (Nikon, Melville, NY) or an Olympus FV-1000 confocal microscope (Olympus, Center Valley, PA). Imaging on the Olympus FV-1000 microscope was performed through use of the VUMC Cell Imaging Shared Resource. The tissue was assessed in a masked fashion.
9
Immunophenotyping of Adherent Cancer Stem Cells
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NACs smeared on slides and adherent CSCs cultured on cover slides that were coated with 0.1% gelatin were fixed while using 4% paraformaldehyde (Wako, Tokyo, Japan) for 20 min., washed two times with PBS, and then blocked using PBS containing 10% FBS at room temperature for one hour. The cells were then incubated overnight at 4 °C with primary antibodies, lineage cell detection cocktail-biotin antibody (Miltenyi Biotec, Bergisch Gladbach, Germany), anti-CD34 antibody (Santa Cruz Biotechnology, Dallas, TX, USA), and anti-CD117/c-kit (Zytomed systems, Berlin, Germany). After incubation, the cells were washed three times with PBS and then incubated with secondary antibodies, Alexa Fluor 555 labeled anti rabbit IgG goat antibody (Thermo Fisher, Waltham, MA, USA), APC labeled anti-Biotin antibody (Miltenyi Biotec, Bergisch Gladbach, Germany), and PE labeled anti-mouse IgG goat antibody (BioLegend, San Diego, CA, USA) for 1 h. Thereafter, they were washed three times with PBS and then mounted with VECTASHIELD® Antifade Mounting Medium with DAPI (Vector Laboratories, Burlingame, CA, USA). The cells were observed with the Olympus FV-1000 microscope (Olympus, Tokyo, Japan).
10
Neuronal Viability Assay for FUS Mutants
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