Cells were cultured on chamber slides and fixed with 4% paraformaldehyde when harvested. Cells were permeabilized and blocked as described earlier for TIFs. The primary antibodies used included gamma-H2AX (Bethyl, A300-081), pRPA32 (Bethyl, A300-245) and 53BP1 (Abcam, ab175933). Secondary antibodies conjugated to an appropriate fluorophore were used to detect the proteins of interest (Invitrogen, A21127, A11001, A11008 and A31572). Cells were counterstained with DAPI to determine the nuclear localization of the protein of interest. Images were acquired with a Zeiss TIRF microscope in a blinded fashion and analyzed with FIJI software.
Tirf microscope
Manufactured by Zeiss
The TIRF (Total Internal Reflection Fluorescence) microscope is an advanced optical imaging system designed for high-resolution visualization of cellular processes and structures. It utilizes the principle of total internal reflection to selectively excite fluorophores within a thin region near the coverslip, enabling precise observation of events occurring at the cell-substrate interface.
Automatically generated - may contain errors
Lab products found in correlation
A21127, by Thermo Fisher Scientific (2 mentions)
A11001, by Thermo Fisher Scientific (2 mentions)
A11008, by Thermo Fisher Scientific (1 mentions)
Ab175933, by Abcam (1 mentions)
Fugene, by Promega (1 mentions)
A21434, by Thermo Fisher Scientific (1 mentions)
Ab6326, by Abcam (1 mentions)
Mitotracker orange cm h2tmros, by Thermo Fisher Scientific (1 mentions)
F1002, by PNA Bio (1 mentions)
Andor ixon 888 emccd cameras, by Oxford Instruments (1 mentions)
Band pass filter, by Zeiss (1 mentions)
12 protocols using tirf microscope
1
Immunofluorescence Microscopy of DNA Damage Markers
2
Quantifying AKT Membrane Recruitment in Live Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were transfected with GFP-AKT-PH and a membrane marker control, either PMT-RFP or Lyn-mCherry. Cells were cultured in 35-mm glass-bottom coverslip dishes (81158, ibidi) and transfected with the relevant constructs using Fugene (Promega). Media was replaced with live-cell imaging solution (HEPES buffered, Invitrogen) and mounted on a TIRF microscope (Zeiss). Cells were illuminated through a 100× objective. Images were acquired every 10 s. Intensities were recorded for 2 min prior to EGF addition. As a control, media was added without EGF. To account for fluctuations in the amount of membrane in the TIRF field, GFP-AKT-PH intensity was divided by relative membrane marker intensity following background subtraction. The relative recruitment of AKT-PH or AKT to the membrane was determined by dividing by the average value prior to EGF addition: where BG denotes background (determined from an area of the image outside the cell body.
3
Telomere Resection Dynamics Visualization
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Actively dividing cells were labeled with CldU for 24 h. Cells were then harvested and embedded in low melting point agarose plugs and digested with proteinase K for 48 h at 37°C. Plugs were then melted and the DNA was combed onto coverslips as described earlier for telomere combing. Combed DNA was fixed and processed in the native state, to allow for the detection of ssDNA and G4s. CldU was detected with a primary antibody (Abcam, ab6326) and AF555-conjugated secondary antibody (Invitrogen, A21434). G4s were detected with a primary antibody (Millipore, MABE1126 and MABE917) and AF488-conjugated secondary antibody (Invitrogen, A11001). Images were acquired with a Zeiss TIRF microscope and processed with Image J/FIJI software. ssDNA fibers were imaged, scored and measured in a double-blind manner. Relative resection was measured in pixels and then normalized.
4
Contractility Analysis of hiPSC-CMs with NLS-IDH2
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
hiPSC-CMs were transduced with NLS-IDH2, and contractility was analyzed 24 h after DRN treatment in comparison with cells not treated with DRN. Beating patterns were captured at 25 frames per second (fps) over 20 s for each position on a Zeiss TIRF microscope with a bright field using a 10X objective. At least 30 positions were captured while maintaining the temperature at 37 °C and CO2 at 5%. The contraction velocity and beating rates were analyzed using the MATLAB-based Conklin motion tracking algorithm52 (link). Briefly, images were divided into arrays of pixel macroblocks, and the motion of each macroblock for subsequent frames was calculated through a single-sweep exhaustive search block matching algorithm. By applying the vector amplitude, motion velocity tracings are generated, featuring typical contraction and relaxation peaks and allowing for the calculation of parameters such as beat rate and peak velocities.
5
Live-cell Imaging of Transferrin Endocytosis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Live mitochondrial staining was achieved by adding 100 nM iron sensor dye RDA (a gift from U. Rauen, Institute of Physiological Chemistry, University Hospital Essen, Essen, Germany) or 100 nM membrane potential-dependent dye MitoTracker orange CM-H2TMRos (Molecular Probes) to the last 15 min of the 1-h preincubation step with imaging media. Then, 50 µg/ml Tf conjugated to AF647 (Invitrogen) or hTf or lock-hTf conjugated to AF488 (Invitrogen) was added to the cells for a 2-min pulse at 37°C. After this, the cells were briefly washed and chased with imaging medium for another 2 min. Starting at time point 5–6 min after Tf pulse, time-lapse videos of 15-s duration were acquired. This entire assay was performed with the dish with cells placed on the oil immersion objective lens inside the temperature-controlled live-cell incubation chamber of the TIRF microscope (ZEISS) to avoid delay in the acquisition of early time points of Tf-endocytic trafficking.
6
Metaphase Chromosome Spread Preparation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Prior to collection, cells were treated with colcemid at 10 mg/ml and incubated for 3 h. After collection, the cells were re-suspended in 75 mM KCl and incubated for 30 min. Cells were then treated with fixative (3:1 MeOH and glacial acetic acid) and dropped over a wet slide at a 45°angle. Slides were subsequently washed with fixative and dried overnight. After drying, the slides were rehydrated, fixed with 4% formaldehyde and then washed and dehydrated in an ethanol series at –20°C. Once dry, the slides were denatured at 80°C and hybridized with a Cy3-conjugated protein:nucleic acid (PNA) probe (C3TA2)3. The slides were then washed, counterstained with DAPI, and dehydrated. Slides were mounted using ProLong Gold. Images were acquired using a Zeiss TIRF microscope. The images were analyzed with FIJI software and scored in a double-blind manner.
7
Telomere Length Analysis in Metaphase Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were pre-treated with colcemid at 10 mg/ml for 3 h prior to collection. Cells were then fixed with methanol and acetic acid at a ratio of 3:1, dropped over a wet slide at a 45° angle and then washed and dried overnight in a fume hood at room temperature. Metaphase-containing slides were then rehydrated with PBS, fixed with 4% paraformaldehyde, dehydrated in a cold ethanol series and heat denatured to allow for hybridization of a Cy3-conjugated PNA telomere probe (C3TA2)3 (PNA Bio, F1002). Slides were then washed and counterstained with DAPI and dehydrated in a cold ethanol series. Images were acquired by a Zeiss TIRF microscope and analyzed with FIJI software. Images were scored in a blind fashion.
8
Telomere Length Quantification by T-FISH
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were grown in 7.5 mM BrdU and 2.5 mM BrdC for 14 h prior to treatment with colcemid at 10 mg/ml for 3 h prior to collection. The cells were then fixed, dropped over a wet slide at a 45° angle and then washed and dried overnight as described for telomere fluorescent in situ hybridization (T-FISH). Metaphase cell-containing slides were then rehydrated in PBS, stained with Hoechst 33258 and exposed to 365 nm UV light. Slides were then digested with ExoIII, washed in PBS and dehydrated in a cold ethanol series. Slides were then individually hybridized with a G- or C-strand-specific PNA probe conjugated to either Cy3 or AF488 (PNA Bio, F1008 and F1002). The slides were then washed, counterstained with DAPI and dehydrated in a cold ethanol series. Images were acquired on a Zeiss TIRF microscope and analyzed with FIJI software. Images were scored in a blind manner.
9
TIRF Microscopy for Viral Capture
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Biotinylated viral particles in HBS (30–100 μL) were flowed through the flow channel and captured on the modified coverslip surface. Unbound viral particles were washed out with 50 μL of HBS pH 7.5. TIRF microscopy assays were carried out with imaging buffer (50 mM HEPES pH 7.0, 100 mM NaCl), which was supplemented with an oxygen quenching system for dual colour experiments to reduce photobleaching (2 mM trolox, 2.5 mM protocatechuic acid, 0.25 U mL−1 protocatechuate-3,4-dioxygenase).
Images were collected on a custom built TIRF microscope based around an ASI-RAMM frame (Applied Scientific Instrumentation) with a Nikon 100 × CFI Apochromat TIRF (1.49 NA) oil immersion objective. Lasers were incorporated using the NicoLase system (Nicovich et al., 2017 (link)). Images were captured on two Andor iXon 888 EMCCD cameras (Andor Technology Ltd). 300 mm tube lenses were used to give a field of view of 88.68 μm × 88.68 μm. Alternatively, images were collected on a TILL Photonics TIRF microscope equipped with a Zeiss 100 × Plan Apochromat (1.46 NA) oil immersion objective, solid state lasers for excitation, a beam splitter for simultaneous dual channel acquisition of fluorescence emission using two Andor iXon 897U EMCCD cameras for detection with a field of view of 46 μm × 46 μm.
Images were collected on a custom built TIRF microscope based around an ASI-RAMM frame (Applied Scientific Instrumentation) with a Nikon 100 × CFI Apochromat TIRF (1.49 NA) oil immersion objective. Lasers were incorporated using the NicoLase system (Nicovich et al., 2017 (link)). Images were captured on two Andor iXon 888 EMCCD cameras (Andor Technology Ltd). 300 mm tube lenses were used to give a field of view of 88.68 μm × 88.68 μm. Alternatively, images were collected on a TILL Photonics TIRF microscope equipped with a Zeiss 100 × Plan Apochromat (1.46 NA) oil immersion objective, solid state lasers for excitation, a beam splitter for simultaneous dual channel acquisition of fluorescence emission using two Andor iXon 897U EMCCD cameras for detection with a field of view of 46 μm × 46 μm.
10
Quantitative Imaging of Islet Cell Cytoskeleton
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The islet cells were fixed at 15 days of culture and labeled with anti-vinculin antibodies, FITC-phalloidin and DAPI. Random fields were imaged by epifluorescence (actin and DAPI) and TIRF (vinculin) microscopy using a Carl Zeiss TIRF microscope, equipped with an Argon laser, using a 100 × 1.45 numerical aperture (NA) oil immersion objective. Green fluorescence was excited using the 488-nm laser line and imaged with a band-pass filter (Zeiss) mounted on a Retiga SRV CCD camera. The images were analyzed using an existing Image-Pro Plus plug-in (object analysis) for selecting and quantifying fluorescent clusters according to their shape, size and fluorescence intensity. For each object, the following parameters were measured in a software-assisted manner: the area (μm2), major axis (μm), minor axis (μm) and the number of vinculin-positive clusters per cell; the length (μm) of actin fibers; the nuclear area (μm2) and aspect (Major axis/minor Axis, where 1.0 corresponds to a perfect circle).
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!