Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was performed using a commercial apoptosis detection kit (Roche, Italy) according to the manufacturer's suggestions. Breafly, treated cells were washed in PBS, fixed in 4% Paraformaldehyde (PFA), and permabilized with 0.1% sodium citrate, 0.1% Triton X-100 in 1xPBS for 2 minutes at 4°C. Nuclei were stained with DAPI (Vector Laboratories, CA, USA) and slides were mounted with Vectashield mounting medium for fluorescence (Vector laboratories). Fluorescence signals were captured at 40X magnitude by using a Leica DMIRE2 microscope equipped with a Leica DFC 350FX camera and elaborated by a Leica FW4000 deconvolution software (Leica, Germany). The percentage of apoptotic cells was calculated by the ratio of TUNEL-positive nuclei and total nuclei stained with DAPI. Quantitative detection of apoptotic cells was also performed on 1×106 cells using TiterTACS colorimetric apoptosis detection kit (Trevigen, MD, USA) according to the manufacturer's instructions.
Fw4000 deconvolution software
Manufactured by Leica
Sourced in Germany
The Leica FW4000 is a deconvolution software designed for microscopy imaging. It is used to enhance image clarity and resolution by removing the blurring effects of the optical system.
Automatically generated - may contain errors
Lab products found in correlation
Dfc350 fx camera, by Leica (5 mentions)
Dmire2 microscope, by Leica (5 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)
Vectashield mounting medium for fluorescence, by Vector Laboratories (1 mentions)
Titertacs colorimetric apoptosis detection kit, by Bio-Techne (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Bio-Rad (1 mentions)
Anti γh2ax, by Merck Group (1 mentions)
Rabbit pab anti trf1 n19, by Santa Cruz Biotechnology (1 mentions)
Anti rabbit igg h l f ab 2 fragment alexa fluor 555 conjugate, by Cell Signaling Technology (1 mentions)
Rabbit pab anti hmgb1, by Abcam (1 mentions)
F ab 2 fragment alexa fluor 488 conjugate, by Cell Signaling Technology (1 mentions)
6 protocols using fw4000 deconvolution software
1
Quantitative Apoptosis Assay using TUNEL
2
Immunofluorescence Staining of YAP and p53
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Cells were fixed in 4% formaldehyde for 20 min at room temperature. Cells were then washed with PBS twice, permeabilized in 0.3% Triton X-100 in PBS for 5 min and blocked in PBS/0,5% BSA for 60 min at room temperature. After cells were incubated overnight at 4 °C with anti-YAP (1:150, G-6, cat. no. sc-376830, Santa Cruz Biotechnology) or with anti-p53 (1:50, DO-1, cat. no. sc-126, Santa Cruz Biotechnology) both diluted in PBS/1% BSA. Next day, Alexa Fluor 488-labeled goat anti-mouse (1:250, cat. no. A11001, Life Technologies) and goat-anti-rabbit (1:250, cat. no. A11008, Life Technologies) for YAP and p53 respectively, were added as secondary antibodies for 2 h at room temperature. DAPI (Bio-Rad) was used for nuclear counterstain for 15 min at room temperature. Images of representative cells for each labeling condition were captured at ×40, ×64, and ×100 magnitudes with a Leica DMIRE2 deconvolution microscope equipped with a Leica DFC 350FX camera and elaborated by FW4000 deconvolution software (Leica, Wetzlar, Germany). The experiments were performed in triplicates.
3
Immunofluorescence and FISH Analysis of PML and TRF1
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Cells were fixed in 2% formaldehyde and permeabilized in 0.25% Triton X-100 in PBS for 5 min at room temperature. For immunolabeling, cells were incubated with anti-PML (goat polyclonal N19 Santa Cruz Biotechnology) and anti-TRF1 (rabbit polyclonal N19, Santa Cruz Biotechnology, Dallas, TX, USA) primary antibodies at room temperature for 2 h, then washed in PBS and incubated with the FITC-conjugated bovine anti-goat (Santa Cruz Biotechnology, Dallas, TX, USA) followed by cy3-conjugated donkey anti-rabbit secondary antibodies (Jackson Immunoresearch). For FISH analysis, cells fixed as above were dehydrated and hybridized with Cy3-coupled PNA telo probe (Panagene) as described by Lenain et al. (2006). Images were captured with an X100 objective. Fluorescent signals were recorded by using a Leica DMIRE2 microscope equipped with a Leica DFC 350FX camera and elaborated by a Leica FW4000 deconvolution software (Leica, Solms, Germany). This system permits to focus single planes inside the cell, generating 3D high-resolution images. For qFISH analysis, telomeric spots intensity was calculated by ImageJ software on images acquired at the same exposure time.
4
Immunofluorescence and FISH Analysis of BPBA-Induced DNA Damage
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HeLa and U2OS cells were seeded and treated with BPBA . At each endpoint, cells were fixed in 2% formaldehyde, permeabilized in 0.25% Triton X-100 in PBS for 5 min at r.t., and incubated with the mouse mAb anti-γH2AX (Millipore, Burlington, MA, USA) followed by the secondary Alexa 488 goat antimouse antibody. Lastly, nuclei were counterstained with DAPI (Sigma-Aldrich, St. Louis, MO, USA). For combined FISH experiments, after immunofluorescence, samples were refixed in 2% formaldehyde and dehydrated by ethanol series. Then, slices were hybridized with telomere-specific (TTAGGG)n-Cy3 PNA probe (Panagene, Daejeon, South Korea) according to the manufacturer’s instruction. Lastly, samples were counterstained with DAPI (Sigma-Aldrich, St. Louis, MO, USA). Fluorescence signals were recorded by using a Leica DMIRE2 microscope equipped with a Leica DFC 350FX camera and elaborated by a Leica FW4000 deconvolution software (Leica, Solms, Germany) at 63× magnification. TIFs images were acquired with a Zeiss LSM confocal laser scanner (Zeiss, Jena, Germany) at 63× magnification.
5
Autophagy Monitoring by LC3B Puncta
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Cells were grown on glass coverslips, treated according to the experiment, fixed in 4% formaldehyde in PBS for 10 minutes at Room Temperature (RT) and permeabilized with 0.25% Triton X-100 for 5 minutes at RT.
For autophagy experiments, cells were transiently transfected with EGFP-LC3B, as reported above, treated for 24 hours with the indicated drugs and then processed for fluorescence experiments. Autophagosome structure formation was detected by observing LC3B puncta in EGFP-LC3B expressing cells. For each experimental condition, at least 150 cells were counted; cells with more than 10 puncta were considered positive for autophagy. The results were represented as the percentage of autophagy positive cells respect EGFP-LC3B expressing cells. Nuclei were stained with DAPI. Fluorescence signals were recorded by using a Leica DMIRE2 microscope equipped with a Leica DFC 350FX camera and elaborated by Leica FW4000 deconvolution software (Leica, Wetzlar, Germany).
For autophagy experiments, cells were transiently transfected with EGFP-LC3B, as reported above, treated for 24 hours with the indicated drugs and then processed for fluorescence experiments. Autophagosome structure formation was detected by observing LC3B puncta in EGFP-LC3B expressing cells. For each experimental condition, at least 150 cells were counted; cells with more than 10 puncta were considered positive for autophagy. The results were represented as the percentage of autophagy positive cells respect EGFP-LC3B expressing cells. Nuclei were stained with DAPI. Fluorescence signals were recorded by using a Leica DMIRE2 microscope equipped with a Leica DFC 350FX camera and elaborated by Leica FW4000 deconvolution software (Leica, Wetzlar, Germany).
6
Quantification of DNA Damage and Telomere Integrity
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Cells were fixed in 2% formaldehyde in phosphate buffered saline (PBS) for 10 min at room temperature (RT) and permeabilized in 0.25% Triton X-100 in PBS for 5 min at RT. For immune-labeling, cells were incubated with primary antibody for 2 h at RT, washed twice in PBS and finally incubated with the secondary antibodies for 1 h. The following primary antibodies were used: Rabbit pAb anti-HMGB1 (Abcam Ltd, Cambridge, UK), Mouse mAb anti-γH2AX (Millipore, Billerica, MA, USA) and Rabbit pAb anti-TRF1 N19 (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The following secondary antibodies were used: Anti-Mouse IgG (H+L), F(ab’)2 Fragment (Alexa Fluor 488 Conjugate) (Cell Signaling) and Anti-rabbit IgG (H+L), F(ab’)2 Fragment (Alexa Fluor 555 Conjugate) (Cell Signaling). Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, Sigma). Fluorescence signals were recorded by using a Leica DMIRE2 microscope equipped with a Leica DFC 350FX camera and elaborated by Leica FW4000 deconvolution software (Leica, Solms, Germany). For quantitative analysis of γH2AX positivity, 300 cells on triplicate slices were scored and for TIF analysis, 30 γH2AX-positive cells were scored. Cells with at least four co-localizations (γH2AX/TRF1) were considered as TIF-positive. Where reported, cells were incubated with the indicated doses of Braco-19 for 24 h.
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