To detect cell apoptosis in brain, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining was performed according to the manufacturer’s protocol (DeadEnd Fluorometric kit, Promega, WI). Three sections per rat were examined and were photographed in parallel for TUNEL-positive cell counting.
Deadend fluorometric kit
Manufactured by Promega
Sourced in United States
The DeadEnd Fluorometric kit is a laboratory product designed to detect and quantify DNA fragmentation, a hallmark of apoptosis. The kit provides a sensitive fluorometric method for measuring DNA fragmentation in cell samples, enabling researchers to study programmed cell death processes.
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6 protocols using deadend fluorometric kit
1
TUNEL Staining for Apoptosis Detection
2
Apoptosis Evaluation in Kidney
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To evaluate apoptosis in the kidney we used the Promega DeadEnd Fluorometric Kit according to the manufacturers protocol (Promega, Madison, Wisconsin, USA). Pictures were taken with an inverted microscope (Axiovert200, equipped with an ApoTome system and an AxioCam MRm camera. Objectives used: Plan Apochromat 20 ×/ 0.8 NA and EC Plan Neofluar 5 ×/ 0,16 NA. All from Carl Zeiss, Jena, Germany) using Axiovision 4.8 software for acquisition and subsequent image processing (Carl Zeiss, Jena, Germany).
3
Apoptosis Analysis in Dgcr8 Knockout Mice
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To analyse apoptosis in the kidneys of Dgcr8 knockout and littermate control mice we utilized the Promega DeadEnd Fluorometric Kit according to the manufacturers protocol. Pictures were taken with an inverted microscope (Axiovert200, equipped with an ApoTome system and an AxioCam MRm camera. Objective used: Plan Apochromat 20×/0.8 NA. Carl Zeiss) using Axiovision 4.8 (Carl Zeiss).
4
Detecting Endothelial Cell Apoptosis
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TUNEL-positive cells exhibit DNA damage, indicating apoptotic cell death (22 (link)). Apoptosis of ECs in the intimal wall was detected by TUNEL staining, following the manufacturer's protocol (DeadEnd Fluorometric kit; Promega, Madison, WI, USA) and as described in a previous study (20 ). To identify vascular EC apoptosis, the sections were visualized on a fluorescence microscope. To assess the extent of EC apoptosis, six microscopic fields were examined and photographed parallel to TUNEL-positive cell counting.
5
Formalin-Fixed TUNEL Assay on A375 Tumors
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A375 tumor tissues collected from the in vivo efficacy study in the above were fixed in formalin phosphate buffer for one week. Then the tissues were processed to get paraffin embedded sections. TUNEL assay was performed using DeadEnd Fluorometric kit (Promega Corporation, Madison, WI) following manufacturer’s instructions. By the end of the experiment, VECTASHIELD Hard Set mounting medium with DAPI (Vector Lab, Inc., Burlingame, CA) was used to mount the tumor slides and stain the nuclei. The final slides were analyzed immediately under a fluorescence microscope (EVOS FL Cell Imaging System, Thermo Fisher Scientific Inc., NY).
6
Quantifying Apoptosis in Cortical Samples
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Four samples from each group were prepared for terminal deoxynucleotidyl TUNEL staining. Apoptosis was detected using a TUNEL kit according to the manufacturer’s protocol (DeadEnd Fluorometric kit, Promega, WI, USA). Slides were then counterstained with 4′,6-diamidino-2-phenylindole (DAPI), washed, coverslipped with a water-based mounting medium, and sealed with nail polish. Three microscope fields (20×) containing TUNEL-positive cells in the cortex were selected and imaged. The number of TUNEL/DAPI-positive cells was calculated as the mean of the numbers obtained from six images per rat. Counting was performed in a blinded manner.
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