Apoptosis of the thoracic aorta was detected by Promega G3250 DeadEnd™ Fluorometric TUNEL System (WI, USA). Briefly, Proteinase K was used to permeabilize the thoracic aortas. The thoracic aortas were then incubated in the Terminal Deoxynucleotidyl Transferase (TdT) solution for 60 min and 2X SCC was used to stop the reaction. DAPI was used to visualize the nuclei.
Deadend fluorometric tunel system g3250
Manufactured by Promega
Sourced in United States
The DeadEnd Fluorometric TUNEL System G3250 is a kit that allows for the detection and quantification of apoptotic cells. It utilizes the TUNEL (Terminal deoxynucleotidyl transferase dUTP Nick-End Labeling) assay, which labels the fragmented DNA of apoptotic cells. The kit provides the necessary reagents to perform this analysis.
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Lab products found in correlation
A0564, by Agilent Technologies (2 mentions)
Axio observer z1 inverted microscope, by Zeiss (2 mentions)
D21490, by Thermo Fisher Scientific (2 mentions)
Fluoview 1000, by Olympus (2 mentions)
Lsm 880 confocal microscope, by Zeiss (2 mentions)
Dm500b microscope, by Leica (1 mentions)
Sp5 inverted confocal microscope, by Leica (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (1 mentions)
Fluoview software, by Olympus (1 mentions)
9 protocols using deadend fluorometric tunel system g3250
1
Apoptosis Detection in Thoracic Aorta
2
Quantifying Beta Cell Death in Transplanted Murine Kidneys
3
Quantifying Beta Cell Death in Transplanted Murine Kidneys
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Kidneys were excised from transplanted mice, fixed in Bouin’s solution, paraffin-embedded, sectioned, and stained with an antibody to insulin (A0564, DAKO), TUNEL (G3250 DeadEnd Fluorometric TUNEL system, Promega) and DAPI (D21490, Invitrogen). Cells doubly positive for insulin and TUNEL staining were quantified and compared to total number of insulin positive cells using fluorescence microscopy. An average of 1166±163 cells were counted per condition and are pooled means of a total of 32 individual mouse grafts with Veh n = 6, RO0281675 n = 5, BAD SAHBA SD n = 6, BAD SAHBA SD + PAA n = 4 and BAD SAHBA AAA n = 4 mice and reported as % β-cell death representing the %TUNEL positive cells normalized to vehicle set to 100%. Imaging was performed at 20X magnification with a CARL ZEISS AXIO Observer Z1 Inverted Microscope using ZEN 2 core imaging Software for Zeiss microscope.
4
Apoptosis Quantification via TUNEL
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To measure the end-stage apoptosis, TUNEL staining on lacrimal gland was performed at different time points of duct ligation. The frozen sections were incubated with 4% paraformaldehyde for twenty minutes, followed by three times PBS washed for five minutes each. Then all sections were covered with 20 µg/mL proteinase K for 15 minutes at room temperature. After three washes with PBS for five minutes each, they were incubated with reagent mix (DeadEnd Fluorometric TUNEL System G3250; Promega, Madison, WI, USA) at 37°C for one hour according to manufacturer's instructions. Then saline sodium citrate were used to terminate reaction for 15 minutes. Tissues were rinsed three times in PBS and counterstained with DAPI, mounted, and photographed with laser confocal microscope (Olympus Fluoview 1000).
5
Quantifying Apoptosis via TUNEL Staining
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To visualize apoptosis, TUNEL staining was performed according to the manufacturer’s instructions using the Promega DeadEnd™ Fluorometric TUNEL System # G3250 [14 (link), 22 (link)]. The fluorescein-12-dUTP-labeled DNA was visualized by Leica DM500 B Microscope and digital camera. DAPI nuclear counterstaining was performed to visualize total cells. Osteocyte and PDL apoptotic fluorescent cells were analyzed using a custom ImageJ macro. Raw images were converted to binary images using the Intermodes threshold, and individual nuclei were counted using the built in Analyze Particles function. TUNEL-positive cells were counted on the compression side of alveolar bone and PDL (mesial surface of the distobuccal root) and normalized per number of DAPI-positive cells in the same tissue section. The ratio of cells was calculated by one observer for each section as TUNEL-positive cells/total cells in both the AB and PDL. The ratio from three sections was then averaged for each mouse. Sixteen randomly selected measurements were repeated with an intra-class coefficient of r = 0.9998. The averaged values were used to conduct statistical analysis.
6
TUNEL Assay for Detecting Apoptosis
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A TUNEL assay was used to detect cell apoptosis in lacrimal glands. Frozen sections, after fixation, were treated with proteinase K for 10 minutes. Subsequently, negative and positive controls were treated with DNAase for 10 minutes each, followed by washes with PBS. Sections were then incubated with equilibration buffer for 10 minutes at room temperature, followed by reagent mix (DeadEnd Fluorometric TUNEL System G3250; Promega Corporation, Madison, WI, USA) at 37°C for one hour. Tissue specimens were then soaked in saline sodium citrate (DeadEnd Fluorometric TUNEL System, G329A; Promega) for 15 minutes. Slides were mounted with DAPI after washing three times with PBS and imaged with a LSM 880 confocal microscope (Zeiss).
7
TUNEL Assay for Apoptosis Detection
8
Quantifying Apoptosis in Corneal Tissue
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To measure end-stage apoptosis, in situ TUNEL assay was performed on frozen sections (DeadEnd Fluorometric TUNEL System G3250; Promega) according to manufacturer’s instructions. Sections were counterstained with DAPI (Vector, Burlingame, CA, USA), and digital images of three representative areas of the cornea were captured with a Leica microscope (DM2500; Leica Microsystems). The average number of TUNEL+ cells was obtained from triplicate slides per sample. For the positive control, sections were incubated in DNase I before the addition of equilibration buffer, while DDW was used instead of the TdT reaction mix in the negative control.
9
TUNEL Assay for Corneal Apoptosis
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In situ cell apoptosis was determined by the TUNEL (terminal deoxynucleotidyl transferase dUTP nick-end labeling) assay (DeadEnd Fluorometric TUNEL System G3250; Promega, Madison, WI, USA) in corneal frozen sections. Sections were counterstained with 4′, 6-diamidino-2-phenylindole I (Vector), mounted and photo images were taken with a confocal laser scanning microscope (Olympus Fluoview 1000; Olympus, Tokyo, Japan). The images were captured and processed using Olympus Fluoview software (Olympus, Tokyo, Japan).
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