The two strains were observed using a Leica TCS-SP5 microscope and field emission scanning electron microscopy to observe the effect of gene knockout on the phenotype of the strains. The Leica TCS-SP5 microscope (Leica Microsystems, Mannheim, Germany) was used to illuminate the field using a 10x eyepiece and a 20x objective for observation, and the cell morphology was imaged by scanning electron microscopy using the critical point drying sample preparation method, mainly to observe the differences between the WT and ΔAhp strains in terms of shape, size, and the degree of colony aggregation.
Tcs sp5 microscope
Manufactured by Leica
Sourced in Germany, United Kingdom, United States
The Leica TCS SP5 is a confocal microscope designed for advanced imaging applications. It features a multi-channel detection system, allowing for the simultaneous capture of multiple fluorescent signals. The microscope is equipped with a range of laser lines to excite a variety of fluorophores, enabling the visualization of complex biological samples. The TCS SP5 provides high-resolution imaging capabilities, making it a versatile tool for researchers and scientists.
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Lab products found in correlation
Las af software, by Leica (15 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (7 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (6 mentions)
Vectashield, by Vector Laboratories (5 mentions)
Alexa fluor 488 green, by Thermo Fisher Scientific (4 mentions)
Illustrator cs5, by Adobe (4 mentions)
Dm4000b microscope, by Leica (4 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (4 mentions)
Paraformaldehyde (pfa), by Merck Group (4 mentions)
Mitotracker green, by Thermo Fisher Scientific (3 mentions)
Las af lite 4, by Leica (3 mentions)
Alexa fluor 594, by Thermo Fisher Scientific (3 mentions)
Dcfh da, by Thermo Fisher Scientific (3 mentions)
4 6 diamidine 2 phenylindole dihydrochloride dapi, by Merck Group (3 mentions)
Alexa fluor 647 anti mouse b220 antibody clone ra3 6b2, by BioLegend (2 mentions)
Imaris software 9, by Oxford Instruments (2 mentions)
Mitotracker green, by Leica (2 mentions)
Donkey serum, by Merck Group (2 mentions)
Snap hq2 ccd color camera, by Nikon (2 mentions)
Eclipse 90i upright microscope, by Nikon (2 mentions)
290 protocols using tcs sp5 microscope
1
Visualizing Mutant Cell Morphology
2
Quantifying Cellular Oxidative Stress
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Sf21 living cells expressing DmCry or the control SPA1 construct were washed 2 times in PBS (pH 7.4) and incubated in PBS containing 58 μM DCFH-DA (Molecular Probes, Life Technologies, Grand Island, NY, USA) for 10 min in the dark, then exposed to blue light during 5 min and observed immediately either between glass and coverslips with a Zeiss AxioImager.Z1/ApoTome microscope or in an observation chamber with an inverted Leica TCS SP5 microscope. Green fluorescence from DCFH-DA was excited at 488 nm. Zeiss AxioImager.Z1/ApoTome observations were done by using a 10X objective. Emission fluorescence intensities were detected by using the Zeiss filter set 38 Endow GFP shift free; EX BP 470/40, BS FT 495, EM BP 525/50 and differential interference contrast (DIC) with an Analy DIC TransLight. The images were digitally captured with a CCD-camera (AxioCam MRm) using the software Axiovision (version 4.7.2, Carl Zeiss).
Inverted Leica TCS SP5 microscope observations were done by using a 40x objective. Emission fluorescence intensities were detected by using a photomultiplicator between 498 and 561 nm and DIC by using a transmission photomultiplicator. Z series projections were performed using ImageJ software (W. S. Rasband, ImageJ).
Inverted Leica TCS SP5 microscope observations were done by using a 40x objective. Emission fluorescence intensities were detected by using a photomultiplicator between 498 and 561 nm and DIC by using a transmission photomultiplicator. Z series projections were performed using ImageJ software (W. S. Rasband, ImageJ).
3
Quantifying Peripheral Sensitization Markers
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The expression of peripheral sensitization-related molecules such as CGRP, TRPV1, NGF, TrkA, and of the NPY was evaluated in DRG sections, from 5 animals in each group, by immunohistochemistry analysis. After deparaffinization, rehydration, antigen retrieval and quenching of endogenous fluorescence, histological sections were blocked at RT, and then incubated with rabbit anti-CGRP (1:6,000, Sigma-Aldrich, USA), or rabbit anti-TRPV1 (1:200, Antibodies-online.com, Aachen, Germany), or rabbit anti-TrkA (1:100, Abcam, UK) or rabbit anti-NPY (1:1,000, Sigma-Aldrich, Germany) overnight at 4 °C. For signal detection, tissue sections were incubated for 1 h at RT with anti-rabbit Alexa Fluor 568 antibody (1:1,000, Life Technologies, USA), incubated with DAPI for the nuclei staining and then mounted with Fluoromount Aqueous Mounting Medium (Sigma-Aldrich, USA). Images were acquired on the confocal Leica TCS SP5 microscope (Leica Microsystems, Germany). The staining intensity was quantified using NIH ImageJ software.
4
Visualization of IL-21 and IL-2 Expression
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Tissues were harvested after perfusing Il21-mCherry/Il2-emGFP TG mice with 4% paraformaldehyde (PFA, pH 7.4 at 4 °C), tissues were further fixed in 4% PFA at 4°C for 2 hours, placed in 30% sucrose for 24 hours, and embedded in OCT compound (Tissue-Tek) for freezing. 10-µm thick frozen tissue sections were rehydrated, blocked with 10% rat serum in 0.1% Tween20/PBS (PBST) for 1 hour at room temperature, and stained with the Alexa Fluor 647 anti-mouse B220 antibody (clone RA3–6B2, Biolegend) in 1% rat serum/ 0.1% PBST overnight at 4 °C. Nuclei were stained with 4’,6-diamidin-2-fenilindolo (DAPI). Images were acquired using a Leica TCS SP5 microscope (Leica Microsystems, Mannheim, Germany) using a 20x objective NA .7, zoom 1X and processed with Imaris software 9.0.0 (Bitplane AG, Zurich Switzerland).
5
Mitochondrial Imaging and Analysis
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Cells were seeded on coverslips and treated with the inhibitors for indicated time, then stained with Mitotracker Orange and Mitotracker Green (Invitrogen, M7510 and M7514) as described by manufacturer. The intensity of fluorescence was visualized using Leica TCS SP5 microscope (Leica Microsystems) at following wavelengths: Ex/Em=577/599 nm for Mitotracker Orange, 490/516 nm for Mitotracker Green. Images were analyzed using ImageJ software.
6
Quantitative Biofilm Imaging by CLSM
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Subsequently, the stained biofilms were analyzed by confocal laser scanning microscopy using a Leica TCS SP5 microscope (Leica Microsystem, Wetzlar, Germany) with x 63/1.4 NA oil-immersion objective lens. Excitation of the dyes were carried out using the following wavelengths: UV laser 405 nm, Argon laser 488 nm and Helium-Neon laser 633 nm. Fluorescence emission was detected at the following wavelengths: 420-460 nm (SYTO 40), 515-570 nm (TOTO-1), and 687-770 nm (DRAQ7). The biofilms were scanned in sequential mode, and z-series were generated by vertical optical sectioning using a step size of 0.5 µm. Image acquisition was conducted in 6x line average mode. Each disc was measured at three different positions. Images were processed using IMARIS software (version 9.7.2, Bitplane, Zurich, Switzerland) and quantified by Fiji. The 3D data were collapsed into 2D by maximal z-projection and each channel was analyzed separately. Contrary to TOTO-1, the threshold of SYTO 40 and DRAQ7 stains was not modified. The measurements were set areas, min/max, gray value, area fraction, mean gray value and stack position and the size of the analyze particles were from 0.10 - infinity micron2.
7
Visualizing NK Cell-Mediated Killing of Tumor Spheroids
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Co-culture of OC spheroids and HSPC-NK cells was performed as described above. For confocal experiments, SKOV-3-GFPluc spheroids were co-cultured with CD56-APC and GAM-AF647 labeled NK cells. After co-culture, spheres were collected, washed mildly with PBS/BSA buffer, and placed in an Ibidi µ-slide eight-well plate (Ibidi, 80826) in RPMI without phenol red, and Propidium idodide (PI) (Sigma-Aldrich, 247–081–0) was added to a final concentration of 50 µg/mL to detect cell death. Imaging was performed with a Leica TCS SP5 microscope (Leica Microsystems, Germany) at 37°C using an HC PL Fluotar 20.0 × 0.5 dry lens. Laser power, gain and offset were kept constant between experiments and conditions. End point experiments were performed at 0, 1, 3, and 5 h and z-stacks of confocal sections were acquired with a step size of 5 µm. Time lapse movies were made by imaging with a time interval of 30 s and a maximum acquisition time of ∼5 h in a fixed z-plane. GFP and PtdIns were excited with the argon laser line at 488 nm and emission was detected between 495 and 550 nm for GFP and between 595 and 640 nm for PtdIns. In a sequential scan APC and GAM-AF647 were excited with the HeNe laser at 633 nm, and emission was measured between 640 nm and 710 nm.
8
Visualizing Intracellular Dynamics of SPIONs
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For confocal microscopy, MCF-7 cells were cultured directly on 12 mm glass-bottom culture dishes (Thermo Scientific, Nunc). The fluorescent stain LysoTracker Red DND-99 (100 nM) was used to label acidic organelles, and DAPI nucleic acid stain (diluted 1:200; both from Molecular Probes, Life Technologies) to label the nucleus. Dyes were added to cell cultures ~30 min before video microscopy.
Samples were washed three times with PBS and the video-microscopy experiment began when 0.25 mg ml−1 SPION were added to cell medium. Fluorescence, light reflection (SPION back-scattering of light) and differential interference contrast (DIC) images of 4 confocal planes on a Leica TCS SP5 microscope (Leica Microsystems) with a 37 °C incubation system were taken every 2.5 min for the 3 h incubation with SPION. Videos were obtained by combining distinct sequential images (z axis projection merge of the 4 plane images) using LAS AF v.2.3.6 software (Leica Microsystems). For single image analyses, we used ImageJ software [25 (link)].
Samples were washed three times with PBS and the video-microscopy experiment began when 0.25 mg ml−1 SPION were added to cell medium. Fluorescence, light reflection (SPION back-scattering of light) and differential interference contrast (DIC) images of 4 confocal planes on a Leica TCS SP5 microscope (Leica Microsystems) with a 37 °C incubation system were taken every 2.5 min for the 3 h incubation with SPION. Videos were obtained by combining distinct sequential images (z axis projection merge of the 4 plane images) using LAS AF v.2.3.6 software (Leica Microsystems). For single image analyses, we used ImageJ software [25 (link)].
9
Automated Microscopy for Parasite Analysis
10
Quantifying Mitochondrial Function in PD-MSCs
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PD-MSCs were stained with MitoSox Red and MitoTracker Green (Invitrogen Corporation) to quantify mitochondrial superoxide production and mitochondrial content, respectively. PD-MSCs (1.3 × 104 cells/well) were seeded into 24-well culture plates and washed with Hanks’ balanced salt solution (HBSS). The plates were incubated with 3uM MitoSox Red (Invitrogen Corporation) and 100 nM MitoTracker Green (Invitrogen Corporation) for 40 min at 37 °C. Cells were then washed with HBSS and incubated with 1 μg/ml diamidino-phenylindole hydrochloride (DAPI; Sigma Aldrich) for 1 min at RT. Fluorescence images were obtained using a confocal microscope (Leica TCS SP5 microscope; Leica microsystems, Wentzler, German, × 100 magnifications). Positive signals of targeted fluorescence were marked by an arrowhead in our data.
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