Mitochondrial mass was determined by the fluorescent probe Mito-tracker Green (Beyotime, Jiangsu, China). Cells were incubated with 100 μM CoCl2 for 24 h after transfection, then incubated with the 100 nM Mito-tracker Green in pre-warmed DMEM culture media for 30 min at 37 °C in the dark. Fluorescence intensity was analyzed by Guava easyCyte 8HT flow cytometry (Merck Millipore, Bille-rica, MA, USA). In addition, cells were also incubated with a culture medium containing 100 nM Mito-tracker Green probe at 37 °C for 30 min, then were incubated with 5 μM Hoechst 33,342 (Sigma, St. Louis, USA) at 37 °C for 30 min before monitored under a Leica TCS SP confocal microscope (Leica Microsystems, Heidelberg, Germany).
Leica tcs sp confocal microscope
Manufactured by Leica Microsystems
Sourced in Germany
The Leica TCS SP confocal microscope is a high-performance imaging system designed for advanced microscopy applications. It features a scanning laser module that allows for the acquisition of high-resolution, optical sectioning images of samples. The microscope is equipped with a range of detection channels and can be configured with various laser lines to accommodate various fluorescent probes. The Leica TCS SP confocal microscope is a versatile tool for researchers and scientists in various fields, including biology, materials science, and nanotechnology.
Automatically generated - may contain errors
Lab products found in correlation
Mitotracker green, by Merck Group (1 mentions)
Hoechst 33342, by Merck Group (1 mentions)
Mito tracker green, by Beyotime (1 mentions)
Alexa fluor 488 goat anti rabbit igg antibody, by Thermo Fisher Scientific (1 mentions)
Lab tek 2 chamber slide, by Thermo Fisher Scientific (1 mentions)
Paraformaldehyde (pfa), by ProSciTech (1 mentions)
Streptavidin cy3, by Merck Group (1 mentions)
8 chamber slide, by BD (1 mentions)
Saponin, by Merck Group (1 mentions)
Biotinylated donkey anti sheep antibody, by Jackson ImmunoResearch (1 mentions)
Fc receptor blocking antibody, by BD (1 mentions)
Clone pc10, by Agilent Technologies (1 mentions)
Lipofectamin 2000, by Thermo Fisher Scientific (1 mentions)
Leica application suite, by Leica camera (1 mentions)
Las af lite software, by Leica Microsystems (1 mentions)
Leica dmi 4000b inverted microscope, by Leica Microsystems (1 mentions)
Poly prep slides, by Merck Group (1 mentions)
Fitc cholera toxin b subunit, by Merck Group (1 mentions)
8 protocols using leica tcs sp confocal microscope
1
Mitochondrial Mass Assessment Methods
2
Immunofluorescence of Egr-1 and pERK-1/2 in 16-HBE cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
16-HBE cells were cultured on Lab-Tek-II chamber slides (four or eight well; Thermo Fisher Scientific) and treated with CSE, fixed with 4% paraformaldehyde in PBS for 20 min at 4°C and incubated with rabbit anti-Egr-1 or phospho-ERK-1/2 antibody (Abcam) at 4°C overnight, followed by Alexa Fluor 488 goat anti-rabbit IgG antibody (Thermo Fisher Scientific). Fluorescence was detected with a Leica-TCS-SP confocal microscope (Leica Microsystems, Wetzlar, Germany) and the accompanying software Leica v2.6.1 (Leica Microsystems).
3
Visualizing TGF-β Receptor Internalization
4
Visualizing CLIC1 and RhoA in BMDCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Resting BMDCs or BMDCs 5 min after initiation of synchronised phagocytosis were fixed with 4% paraformaldehyde (Cat #C004, ProSciTech) on 8-chamber slides (Cat #354108, BD Biosciences), then permeabilised with 0.05% saponin (Cat #S4521, Sigma-Aldrich) (Jiang et al., 2012 (link)). After blocking with 2% IgG free BSA (Cat #001-000-161, Jackson ImmunoResearch Labs) and 1 µg/ml Fc receptor blocking antibody (Cat #553142, BD Biosciences), the cells were stained for; CLIC1 and RhoA. Briefly, BMDCs were firstly stained with 1:100 rabbit anti-mouse RhoA antibody then 1:100 donkey anti-rabbit Cy2 antibody (Cat#ab6940, Abcam) followed by an in-house-derived 1:1000 sheep anti-mouse CLIC1 (Jiang et al., 2012 (link)), then 1:100 biotinylated donkey anti-sheep antibody (Cat #713-065-003, Jackson ImmunoResearch Labs) and finally streptavidin Cy3 (Cat#S6402, Sigma-Aldrich) (Jiang et al., 2012 (link)). Confocal images were obtained on a Leica TCS SP confocal microscope (Leica Microsystems, Germany) and processed using ImageJ64 (NIH, imagej.nih.gov/ij/download/).
5
Immunocytochemistry and RNA ISH in Larvae and Adult Brains
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immunocytochemistry was performed after RNA ISH on larvae or on adult brain sections following a previously described protocol (Yamamoto et al., 2011 (link)). Primary monoclonal anti-Tyrosine Hydroxylase (TH, clone LNC1, MERCK Millipore, Darmstadt, Germany) mouse antibody purified from PC12 cells was used to identify catecholaminergic neurons (Yamamoto et al., 2011 (link)). To identify proliferating cells a monoclonal anti-proliferating cell nuclear antigen (PCNA, Clone PC10, Dako, Glostrup, Denmark) mouse antibody was used (Mueller and Wullimann, 2015 ). As secondary antibody the donkey anti-mouse Alexa-488 IgG (H + L) antibody (Jackson ImmunoResearch Labratories, Suffolk, UK) was applied. Primary antibodies were used in a dilution of 1:100, the secondary antibody in a dilution of 1:500. Microscopy images were obtained with a Leitz Aristoplan or a Leica TCS SP confocal microscope (Leica Microsystems GmbH, Wetzlar, Germany).
6
Colocalization of TβR-II and Caveolin-1
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The Mv1Lu cells were placed in a 24-mm coverglass and transiently transfected with TβR-II-HA plasmid (0.4 μg) by using lipofectamin 2000 (Invitrogen) in accordance to the manufacturer protocol. The transfected cells were pretreated with 5 μg/mL of BetA at 37 °C for 1 h, and then incubated with 100 pM of TGF-β1 for 30 min. After TGF-β1 stimulation, the cells were fixed in methanol at −20 °C for 15 min, washed with PBS, and then blocked using 0.2 % gelatin in PBS for 1 h. Cells were incubated overnight at 4 °C in a humidified chamber with a goat antibody against HA-probe (F-7, Santa Cruz Biotechnology) and a rabbit antibody against caveolin-1 (N-20, Santa Cruz Biotechnology) at 1:100 dilutions. After extensive washing, the cells were incubated with Rhodamine-conjugated donkey anti-goat antibody and FITC-conjugated mouse anti-rabbit antibody at a 1:50 dilution for 1 h. Images were acquired using a Leica TCS SP confocal microscope (Leica Microsystems Ltd., Heidelberg, Germany). The measurements of the colocalization rate were analyzed using a Leica Application Suite.
7
TGF-β1 Signaling and Caveolin-1 Colocalization
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mv1Lu cells transiently expressing TβR-II-HA [37 (link)] were grown on coverslips overnight (50% confluency) and pretreated with 5 μg/ml euphol at 37°C for 1 hour and then stimulated with 100 pM TGF-β1 for 30 minutes. After TGF-β stimulation, cells were fixed in methanol at -20°C for 15 minutes, washed with PBS and then blocked by 0.2% gelatin in PBS for 1 hour. Cells were incubated overnight at 4°C in a humidified chamber with a mouse antibody against hemagglutinin (HA) protein (F-7; Santa Cruz Biotechnology) and rabbit antibody against caveolin-1 (N-20; Santa Cruz Biotechnology) at 1:100 dilution. After extensive washing, cells were incubated with Rhodamine-conjugated donkey anti-mouse antibody and FITC-conjugated goat anti-rabbit antibody at a 1:50 dilution for 1 hour. Images were acquired using a Leica TCS SP confocal microscope (Leica Microsystems Ltd., Heidelberg, Germany). The measurements of colocalization rate were analyzed using a Leica Application Suite.
8
Immunofluorescence Assay for Lipid Raft Visualization
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For immunofluorescence assays, cells were immobilized in PolyPrep slides (Sigma-Aldrich) for 15 min and then fixed with 2% paraformaldehyde (PFA)–0.025% glutaraldehyde in 1× PBS for 10 min at room temperature. After washing twice with 0.1% glycine/PBS, cells were permeabilized with 0.1% Triton X-100/PBS. Incubation with each primary and secondary antibodies and subsequent washes were performed with 1× PBS–2% BSA–0.05% saponine buffer. 4′,6-diamidino-2-phenylindole (Dapi) was used for nuclear staining while cholera toxin B subunit FITC (fluorescein isothiocyanate) conjugate was used as marker of membrane lipid rafts (Sigma-Aldrich). Images were obtained with Leica TCS-SP confocal microscope or Leica DMI 4000B Inverted Microscope (Leica Microsystems, Wetzlar, Germany). The intensity mean per pixel was calculated in confocal images using LAS AF Lite software (Leica Microsystems), and values were represented in bar diagrams showing statistical significance.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!