Differentiated hNECs were processed as previously described (Wong et al., 2022c (link)). Briefly, samples were fixed in 4% paraformaldehyde for 15 min at room temperature. Fixed samples were permeabilized with 0.5% Triton-X in PBS for 10 min on ice and blocked using IF buffer (0.1% BSA, 0.2% Triton and 0.05% Tween 20 in PBS) with 10% normal goat serum (Sigma G9023) for 1 h at room temperature. Samples were incubated with Acetyl-α-Tubulin (Lys40) (D20G3) XP® Rabbit mAb (Alexa Fluor® 647 Conjugate) (Cell Signalling 81502S), Phalloidin-Atto 565 (Sigma 94072) and DAPI (ThermoFisher D1306) for 3 h at room temperature. Membranes were excised from transwells and mounted on SuperFrost Plus slides (Thermo Fisher Scientific, Waltham, MA) with Vectashield Plus antifade mounting medium (H-1900; Vector Laboratories, Burlingame, CA). Images were acquired using a Leica TCS SP8 DLS confocal microscope (Leica Microsystems, Wetzlar, Germany) with a 63x/1.4 oil immersion objective. Image processing was performed using ImageJ software (National Institute of Health, Bethesda, MD).
Tcs sp8 dls confocal microscope
Manufactured by Leica
Sourced in Germany
The Leica TCS SP8 DLS confocal microscope is a high-performance imaging system designed for advanced microscopy applications. It features a tunable supercontinuum laser source that provides a wide range of excitation wavelengths, enabling comprehensive multi-fluorescence imaging. The system is equipped with highly sensitive detectors and advanced optics to capture detailed, high-resolution images of samples.
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Lab products found in correlation
Mitotracker red, by Thermo Fisher Scientific (3 mentions)
Superfrost plus slide, by Thermo Fisher Scientific (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Vectashield antifade mounting medium, by Vector Laboratories (2 mentions)
Normal goat serum (ngs), by Merck Group (1 mentions)
Vectashield plus antifade mounting medium, by Vector Laboratories (1 mentions)
Phalloidin atto 565, by Merck Group (1 mentions)
Nile red, by Merck Group (1 mentions)
Calcofluor white, by Merck Group (1 mentions)
Rabbit anti 53bp1, by Cell Signaling Technology (1 mentions)
Paraformaldehyde (pfa), by ProSciTech (1 mentions)
Application suite advanced fluorescence las af software, by Leica (1 mentions)
Lab tek, by Thermo Fisher Scientific (1 mentions)
Anti mouse alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Anti mouse alexa fluor 647, by Thermo Fisher Scientific (1 mentions)
Fitc bsa, by Merck Group (1 mentions)
Dq red bsa, by Thermo Fisher Scientific (1 mentions)
Anti ar, by Santa Cruz Biotechnology (1 mentions)
Fluorsave reagent, by Merck Group (1 mentions)
Anti vimentin, by Thermo Fisher Scientific (1 mentions)
10 protocols using tcs sp8 dls confocal microscope
1
Immunofluorescent Staining of Differentiated hNECs
2
Comparative Fungal-Bacterial Cell Morphometry
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The allometry of fungal cell types was compared with the marine bacterium Joostella sp. for reference. Fungal cultures were stained with Calcofluor White (Sigma, UK; λ = 360 nm EX/460 nm EM) for cell wall visualisation. Joostella sp. cells were stained with MitoTracker Red (Thermo Fisher, Waltham, MA, USA; λ = 579 nm EX/599 nm EM) for cell membrane visualisation. Both were also stained with Nile Red (Sigma; λ = 552 nm EX/636 nm EM) for qualitative lipid analysis. Images were captured using a Leica TCS SP8 DLS confocal microscope (Leica Microsystems, Germany). For estimations of cell volume, yeast and bacteria cells were treated as a capsule. For filamentous cells, a cylinder was used to calculate volume. In the case of chytrids, rhizoids were discounted from cellular volume calculations and a sphere was used for estimations.
3
Immunofluorescence Imaging of γH2AX and 53BP1
4
Lysosomal Degradation Capacity Assay
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Fat bodies were dissected in Schneider’s Drosophila medium (Biowest, #L0207-500) and immediately pulsed for 10 min at room temperature with a mix of 100 µg ml−1 FITC–BSA (Sigma, #A9771) and 100 µg ml−1 DQ Red BSA (Life Technologies, #D12051 ) in Schneider’s Drosophila medium. Samples were rinsed with PBS and incubated for 2 h (chase) in Schneider’s Drosophila medium. Samples were rinsed with PBS and mounted with VECTASHIELD Antifade Mounting Medium (Vector Laboratories, #H-1000). Three images per sample were captured using a Leica TCS SP8 DLS confocal microscope with a 20× objective and 6× digital zoom-in. Settings of the confocal microscope were kept consistent between images of an experiment. FITC channel images were processed by background subtraction, median filtering and spot quantification using Imaris 9 (Bitplane). FITC-positive puncta were defined as lysosomes and the mean fluorescence intensity of DQ-Red within these lysosomes was quantified as a readout of lysosomal degradation capacity.
5
Confocal Brain Imaging Workflow
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Different systems may have other functions to navigate throughout the slide, such as the Mark&Find or the Tiling tool.troubleshooting 2 and 3 to fix eventual problems with the staining.
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Different systems may have other functions to navigate throughout the slide, such as the Mark&Find or the Tiling tool.
Switch on the software and the laser.
First scan with the navigator system the whole slide with a 2.5× objective to visualize all the brain sections mounted.
Define a region of interest (ROI) around each single section.
Set the 10× objective and redefine the Z-stack of each section, using a 10 μm z-step.
Acquire zoom 1×, bidirectional scanning and resolution set at 1024 × 1024 pixels, frequency 400 Hz.
Z-stack Images are merged and ready to be saved and processed as ".lif” file.
6
Visualizing DNA-GUV Binding
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For DNA–GUV binding, 5 nM of DNA strands were used. DNA binding on GUVs was imaged using a Leica TCS SP8 DLS confocal microscope with HC PL APO CS2 63× oil immersion objective lens, acousto-optical beam splitter and programmable crystal-based beam splitter (Leica Microsystems GmbH, Germany). Two-channel images with rhodamine liposomes (excitation/emission 561/569-611 nm) and Alexa647-DNA (excitation/emission 640/690-734 nm) were imaged with a line averaging of two.
7
Immunohistochemical Analysis of Rat Knee Joints
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Knee joint tissue from all groups were fixed in 4% paraformaldehyde for at least 24 h, and subjected to dehydrated and embedded in paraffin at 60 ℃. 5 μm thick sections were dehydrated, deparaffinized, rehydrated. Sections of rat synovial tissue were double incubated with anti-AR (1:250, Santa Cruz) and anti-Vimentin (1:250, Invitrogen) or anti-4-HNE-protein conjugate (1:250, Invitrogen) primary antibodies at 4 ℃ overnight. Then incubated with fluorescent secondary antibody (anti-rabbit Alexa 555 or anti-mouse FITC, Beyotime) in the dark at room temperature for 2 h. Finally, mounted with FluorSaveTM Reagent (Millipore). Fluorescent images were captured with TCS SP8 DLS confocal microscope (Leica, German). For histological analysis, rat joint sections from all groups were stained with hematoxylin and eosin, and the pictures of stained joint tissues were captured by LED optical microscope (Leica DM2500).
8
Lysosome Quantification via Confocal Imaging
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Tissues were dissected in PBS and stained with LysoTracker Red DND-99 (ThermoFisher, #L7528, 1:2,000) and Hoechst 33342 (Enzo, #ENZ-52401, 0.5 ng µl−1). Samples were rinsed with PBS and mounted with VECTASHIELD Antifade Mounting Medium (Vector Laboratories, #H-1000). Three images per sample were captured using a Leica TCS SP8 DLS confocal microscope with a 20× objective and 6× digital zoom-in. Images were processed by background subtraction, median filtering and spot quantification using Imaris 9 (Bitplane). Settings of the confocal microscope were kept consistent between images of an experiment. LysoTracker-positive puncta were defined as large, when the diameter was greater than 1.5 µm, which is the 75th percentile of the diameter of Rapa-induced LysoTracker-positive puncta.
9
Gut Dysplasia Quantification Protocol
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Guts were dissected in PBS and fixed 30 min with 4% formaldehyde, methanol-free (ThermoFisher, #28908). Samples were washed with PBS and mounted with VECTASHIELD Antifade Mounting Medium with DAPI (Vector Laboratories, #H-1200). The R2 region of the gut was captured using a Leica TCS SP8 DLS confocal microscope with a 40× objective. Images were analyzed using Fiji109 (link) (US National Institutes of Health). The percentage of dysplasia was calculated as the ratio between the length of multilayer nuclei region and the total length of the R2 region of the gut.
10
Retinal Flatmount Immunohistochemistry
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Immunohistochemistry was performed similar to the prior studies (20 (link), 24 (link), 26 (link)). After SHG imaging, the retinal flatmount was fixed with 4% paraformaldehyde for 20 minutes at room temperature. The sample was dehydrated sequentially in 25%, 50%, 75%, and 100% cold methanol for 15 min each and then permeabilized with dichloromethane for 2 hours. Then the retina was rehydrated in 75%, 50%, 25%, and 0% cold methanol for 15 min each. The sample was blocked in a buffer containing 5% normal serum and 0.3% Triton™ X-100 (Thermo Fisher Scientific, Inc.) for 3 hours. The sample was incubated in the primary antibody buffer at 4°C for 3 days. Rabbit and mouse monoclonal antibodies against βIII tubulin (EP1569Y, Abcam, Inc.) and pNF (2F11, EMD MilliporeSigma), respectively, were used at 1:300 dilution. The sample was incubated in the secondary antibody buffer at 4°C for 2 hours. Goat anti-rabbit and anti-mouse IgG antibodies conjugated with Alexa Fluor 594 and Alexa Fluor 647, respectively, were used (AB150080 and AB48389, respectively, Abcam, Inc.). The sample was mounted on a glass slide in Vectashield medium (Vector Laboratories, Inc.), and then imaged by confocal microscopy with Leica TCS SP8 DLS confocal microscope using an oil-immersion objective lens (HC PL APO CS2 40× 1.3NA, Leica).
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