Spinal cord sections prepared as described were fixed, blocked, and incubated with anti-NeuN (1:800, rabbit IgG; Abcam, USA) overnight at 4 °C. The sections were washed with PBS and incubated with Alexa Fluor 488-conjugated goat anti-rabbit IgG (1:200, Jackson ImmunoResearch, West Grove, PA, USA) for 2 h, and then reacted with the TUNEL reaction mixture (Promega) at 37 °C for 1 h. The nuclei were counterstained with DAPI. The proportion of TUNEL-positive neurons in each group of animals was counted under a fluorescence microscope.
Tunel reaction mixture
Manufactured by Promega
Sourced in United States
The TUNEL reaction mixture is a laboratory reagent used for the detection of apoptosis, a type of programmed cell death. It contains the necessary components for performing the Terminal deoxynucleotidyl transferase dUTP Nick End Labeling (TUNEL) assay, which is a widely used technique for identifying and quantifying cells undergoing apoptosis. The mixture includes the essential enzymes, buffers, and labeling reagents required to carry out the TUNEL reaction.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 488 conjugated goat anti rabbit igg, by Jackson ImmunoResearch (1 mentions)
Anti neun, by Abcam (1 mentions)
Ckx53, by Olympus (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Solarbio (1 mentions)
Proteinase k, by Promega (1 mentions)
Lsm 780, by Zeiss (1 mentions)
Superfrost plus slide, by Thermo Fisher Scientific (1 mentions)
Zen 2012, by Zeiss (1 mentions)
6 protocols using tunel reaction mixture
1
Immunohistochemical Analysis of Neuronal Apoptosis
2
TUNEL Assay for Apoptosis Quantification
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To assess apoptosis, the cells were fixed in 4% paraformaldehyde in PBS for 1h at 25°C. After washing in PBS, the cells were permeabilized with 0.1% Triton X-100 in 0.1% sodium citrate for 2min on ice and incubated with 50 μL terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) reaction mixture (Promega) for 1h at 37°C. Cells were then counterstained with DAPI. The number of TUNEL-positive cells was counted using fluorescence microscopy and was normalized to the number of DAPI-positive cells.
3
Gefitinib-Induced Apoptosis Evaluation
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Cells (5 × 104) on coverslips were treated with the indicated concentrations of gefitinib for 48 h, fixed with 4% paraformaldehyde for 25 min and washed twice with PBS. After further washing with PBS, the cells were incubated on ice with 0.1% Triton X-100 in 0.1% sodium citrate for 2 min. The cells were then washed with PBS and incubated with 50 µL TUNEL reaction mixture (cat: G3250; Promega, USA) containing the rTdT enzyme for 1 h at 37°C. After washing three times with PBS, the cells were incubated with 50 µL PI for 15 min in the dark, washed, and analyzed under an inverted fluorescence microscope (CKX53; Olympus, Japan). TUNEL-positive (apoptotic) cells were quantified by counting green-colored cells in 10 fields.
4
TUNEL Assay for Apoptosis Detection
5
Quantifying Liver Tissue Apoptosis
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Paraffin sections of liver tissue were routinely dewaxed, dehydrated and digested by proteinase K (Promega Corp., Madison, WI, USA). Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) reaction mixture (Promega) was added dropwise and incubated at 37°C for 1 h. The average number of apoptotic cells was analyzed in 10 randomly selected visual fields using fluorescence microscopy (magnification, ×200; Nikon Ni-U; Nikon Corp., Tokyo, Japan), and the mean number of apoptotic cells was measured by staining cell nuclei wih DAPI (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) to determine the total number of cells.
6
TUNEL Assay for Apoptosis Detection
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Paraffin-embedded uterine tissues were sectioned at 5-μm intervals and collected on Superfrost Plus slides (Fisher Scientific, Pittsburgh, PA). Paraffin sections were deparaffinized through xylene and rehydrated through an alcohol series. Sections were permeabilized with 20-μg/mL proteinase K for 8-10 minutes at room temperature. The sections were rinsed in phosphate buffered saline and incubated with the TUNEL reaction mixture containing terminal deoxynucleotidyl transferase and labeling solution (nucleotide mixture fluorescein-labeled) (Promega, #G3250, Madison, WI) for 1 hour at 37 °C in a humidified chamber. Sections were mounted and coverslipped using Vectashield that contains DAPI (4′,6-diamidino-2-phenylindole) from Vector Laboratories, #H-1200 (Newark, CA). TUNEL-positive cells were detected using a confocal fluorescence microscope (LSM 780, Zeiss) using a fluorescein filter, and images were processed using ZEN 2012 software (Carl Zeiss Microscopy, LLCOne, White Plains, NY).
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