The freshly purified full-length human Tau protein was incubated with TAMRA (red fluorescence, excitation at 546 nm) at a Tau: TAMRA molar ratio of 1:3 for 1 h. These labeled proteins were filtered and freeze-dried. In total, 5.0, 10.0, 15.0, and 20.0 μM Tau protein labeled by TAMRA were incubated with 10 mM HEPES buffer (pH 7.4) containing 10% (w/v) PEG 4000 and 2 mM β-mercaptoethanol or incubated with the same buffer further containing 10.0 μM myricetin on 25 °C for 5 min to induce LLPS. Liquid droplets of Tau were observed by a Leica TCS SP8 laser scanning confocal microscope with excitation at 546 nm. All phase separation experiments were performed at least three times and were quite reproducible.
Tcs sp8 laser
Manufactured by Leica
Sourced in Germany
The TCS SP8 laser is a versatile and advanced confocal microscope system designed for high-resolution imaging. It features a range of laser excitation sources, enabling researchers to explore a wide variety of fluorescent samples. The system's core function is to provide detailed, high-quality imaging capabilities for various applications in life science research.
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Lab products found in correlation
Las x, by Leica (3 mentions)
Liachroic lightning system, by Leica (2 mentions)
Las x lightning expert, by Leica (2 mentions)
Oil immersion objective, by Leica (2 mentions)
μ slide 8 well chamber, by Ibidi (2 mentions)
Lsm 700, by Zeiss (2 mentions)
Prolong gold antifade mountant with dapi, by Thermo Fisher Scientific (1 mentions)
Volocity 6, by Quorum Technologies (1 mentions)
Dmi6000b widefield microscope, by Leica (1 mentions)
Rhodamine phalloidin, by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Santa Cruz Biotechnology (1 mentions)
Triton x 100, by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (1 mentions)
Hc fluotar l 25x 1.00 imm motcorr objective, by Leica (1 mentions)
Anti sox2, by Cell Signaling Technology (1 mentions)
Anti oct4, by Cell Signaling Technology (1 mentions)
Anti nanog, by Cell Signaling Technology (1 mentions)
Anti dspp, by Santa Cruz Biotechnology (1 mentions)
Anti vimentin, by ZSGB-BIO (1 mentions)
Goat anti rabbit igg rbitc, by Solarbio (1 mentions)
21 protocols using tcs sp8 laser
1
Tau Protein LLPS Assay
2
Lymphatic Valve Anatomy in Mice
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Mesenteries from P3 pups were collected and immunostained with PROX1, FOXC2 and VE-cadherin. High magnification confocal images of lymphangion and valve areas were obtained on a Leica TCS SP8 laser scanning confocal microscope. Using Fiji software, a ROI was drawn around each lymphangion or valve and added to ‘ROI manager’. The area of each ROI was the same for all lymphangion and valve regions. The mean pixel intensity was measured in each ROI in each image. Between 4–6 lymphangion or valve areas were imaged and analyzed from 5–6 control and knockout littermates.
3
Immunofluorescence Staining for R-Loops
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DLD-1 cells were seeded on coverslips at least 24 h before the experiment. After washing with PBS, cells were incubated with 4% paraformaldehyde (PFA) for 10 min. After washing three times with PBS, cells were permeabilized with 0.5% Triton X-100 in PBS for 10 min and blocked with 4% BSA in PBS for 30 min. Primary antibodies were dissolved in ice-cold 4% BSA with the dilution ratio recommended by producers, and the cells were then immersed in the primary antibody buffer for overnight incubation at 4 °C. After three washes in PBS, cells were incubated with the appropriate secondary antibodies for 1 h. Next, cells were mounted in ProLong Gold Antifade Mountant with DAPI (Invitrogen) before imaging. For rapid R-loop immunofluorescence, GFP–RNASEH1 was used as the primary sensor, and the protein was purified as previously described39 . Cells were incubated with 2 μg of GFP–dRNASEH1 in 4% BSA overnight at 4 °C. After washing three times with PBS, cells were directly mounted before imaging. The presented images were obtained using the Leica TCS SP8 laser-scanning confocal microscopy. Unless otherwise indicated, all procedures were performed at room temperature.
4
Microscopic Imaging for Graft Analysis
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Images were captured using either a flatbed scanner Epson Perfection V850 PRO, a Leica DMI6000B widefield microscope or a Leica TCS SP8 laser scanning confocal microscope. The image acquisition software was Leica LAS X and images were processed using Volocity 6.5.1 (Quorum Technologies) and Adobe Photoshop. Any adjustments were applied equally across the entire image, and without the loss of any information. The following images were digitally stitched from multiple images: Figs. 1 b, 2 h, 3 a–f, 4b, c , Supplementary Figs. 1k , 5b and 7d, e, g–j . Figures 1 a and 4a were created by the authors. Each image represents a typical result obtained from analysis of minimum 2–3 graft sections per animal.
5
Characterizing Cell Morphology and ECM Porosity
6
Multicolor Confocal Imaging Protocols
7
Fluorescent Cell Wall Staining Protocol
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For staining with Calcofluor white (CFW), hyphae that incubated on YCS-coated glass slides as mentioned above were stained with a 10-μg ml-1 CFW aqueous solution (Sigma-Aldrich, Merck, USA) for 10 min in the dark. The fluorescence was imaged via the Leica TCS SP8 laser scanning confocal microscope, with excitation of 405-nm UV laser and emission wavelength range of 430–460 nm. The projection of z-stack images was performed with ImageJ (http://rsbweb.nih.gov/ij/ , version 1.47g). Quantification of the fluorescent intensity was performed by measuring the mean gray value using ImageJ software. For each strain, more than 10 hyphae were measured in each experiment, and the entire experiment was conducted twice.
8
Confocal Imaging of CLARITY-Treated Brains
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Brain sections were imaged using a Zeiss LSM700 laser scanning confocal microscope with 20X (numerical aperture (NA) 1.30) and 40X oil immersion (NA 1.25) objective lens (Zeiss). For all confocal images, stacks were acquired using 1 µm‐Z steps. Acquired images were adjusted for brightness and contrast using FIJI/ImageJ software.
CLARITY‐treated brains were mounted in a fructose‐based high refractive index solution (fHRI) prepared as follows: 70% fructose, 20% DMSO in 0.002 M PBS, 0.005% sodium azide. The refractive index of the solution was adjusted to 1.4571 using a refractometer (Krüss GmbH, Hamburg, Germany). In preparation for imaging, samples were incubated in 50% fHRI/50% PBST for 6 hr and finally incubated in 100% fHRI for at least 12 hr. For imaging, samples were mounted in 1% low melting point agarose and covered with fHRI. Whole‐mount brain fluorescence was captured using a Leica TCS SP8 laser scanning confocal microscope equipped with a Leica HC FLUOTAR L 25x/1.00 IMM motCorr objective. Fluorescence signal was detected by exciting the fluorophores with lasers at wavelength of 488 and 552 nm. Detection was performed by two internal photomultipliers (PMT).
CLARITY‐treated brains were mounted in a fructose‐based high refractive index solution (fHRI) prepared as follows: 70% fructose, 20% DMSO in 0.002 M PBS, 0.005% sodium azide. The refractive index of the solution was adjusted to 1.4571 using a refractometer (Krüss GmbH, Hamburg, Germany). In preparation for imaging, samples were incubated in 50% fHRI/50% PBST for 6 hr and finally incubated in 100% fHRI for at least 12 hr. For imaging, samples were mounted in 1% low melting point agarose and covered with fHRI. Whole‐mount brain fluorescence was captured using a Leica TCS SP8 laser scanning confocal microscope equipped with a Leica HC FLUOTAR L 25x/1.00 IMM motCorr objective. Fluorescence signal was detected by exciting the fluorophores with lasers at wavelength of 488 and 552 nm. Detection was performed by two internal photomultipliers (PMT).
9
Multicolor Confocal Imaging Protocols
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12-bit confocal images were acquired on Leica TCS SP8 laser scanning
microscopes. We used 405 nm wavelength for excitation of DAPI; 448 and 458 nm
for excitation of Cerulean; 488 nm for EGFP and Alexa Fluor(AF) 488; 514 nm for
EYFP; 552 and 561 nm for mApple, mScarlet, mCherry, tagRFP, and AF555; 633 nm
for AF633, AF647, Opal650 and BODIPY630/650. For fast confocal live cell imaging
of cell motility and GCaMP transients, we used an 8 kHz resonant scanner. All
other acquisitions were carried out with a Galvo scanner. For overview images
and analysis of cell numbers (i.e. nuclear transgenes and EDU), we used
10x/0.4NA (acquisition with 568nm pixel size (xy), 2 μm z-spacing) and
20x/0.7NA (acquisition with 142nm pixel size (xy), 1 μm z-spacing)
objectives. For analysis of stained cryosections, we used 63x/1.2NA
H2O, and 63x/1.3NA glycerol objectives and acquired images with
at least 100nm pixel size (xy) and 1 μm z-spacing. For all other
analysis, images were acquired using a 25x/0.95NA H2O objective with
114-151nm pixel size (xy) and 1 μm z-spacing. When images were acquired
for subsequent deconvolution, x/y/z parameters were increased closer to Nyquist
resolution to be compatible for processing with Huygens software.
microscopes. We used 405 nm wavelength for excitation of DAPI; 448 and 458 nm
for excitation of Cerulean; 488 nm for EGFP and Alexa Fluor(AF) 488; 514 nm for
EYFP; 552 and 561 nm for mApple, mScarlet, mCherry, tagRFP, and AF555; 633 nm
for AF633, AF647, Opal650 and BODIPY630/650. For fast confocal live cell imaging
of cell motility and GCaMP transients, we used an 8 kHz resonant scanner. All
other acquisitions were carried out with a Galvo scanner. For overview images
and analysis of cell numbers (i.e. nuclear transgenes and EDU), we used
10x/0.4NA (acquisition with 568nm pixel size (xy), 2 μm z-spacing) and
20x/0.7NA (acquisition with 142nm pixel size (xy), 1 μm z-spacing)
objectives. For analysis of stained cryosections, we used 63x/1.2NA
H2O, and 63x/1.3NA glycerol objectives and acquired images with
at least 100nm pixel size (xy) and 1 μm z-spacing. For all other
analysis, images were acquired using a 25x/0.95NA H2O objective with
114-151nm pixel size (xy) and 1 μm z-spacing. When images were acquired
for subsequent deconvolution, x/y/z parameters were increased closer to Nyquist
resolution to be compatible for processing with Huygens software.
10
Immunofluorescence Characterization of Stem Cell Spheroids
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After 4 d, spheroid formation or monolayer on slides was washed with PBS and fixed in 4% paraformaldehyde at room temperature for 10 min, after three times washed with PBST, and permeabilised with 0.1% Triton X-100 solution for 15 min at room temperature. Then, washed with PBST, primary antibodies were then incubated with samples overnight at 4°C: anti-Oct4 (1:200, Cell Signaling Technology), anti-Sox 2(1:200, Cell Signaling Technology), anti-Nanog (1:200, Cell Signaling Technology), anti-CK14 (1:200, ZSGB-BIO, Beijing, China), anti-Vimentin (1:200, ZSGB-BIO), anti-DSPP (1:100, Santa Cruz Biotechnology) and anti-DMP-1 (1:100, Abcam). After incubation with primary antibodies, samples were washed with PBST and then incubated with goat anti-rabbit IgG/RBITC or goat anti-mouse IgG/RBITC (1:100, Solarbio Life Science, Beijing, China) for 1 h at room temperature. For nuclear DNA dye, the samples were mounted with DAPI. Images were captured using the Leica TCS SP8 laser scanning confocal microscopy system.
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