Frozen renal specimens underwent an initial blocking process. Subsequently, sections were subjected to overnight incubation with primary antibodies, including IgG-FITC (Abcam, MA, USA), C3 (MP Biomedicals, CA, USA), TGF-β1 (CellSignalingTechnology, MA, USA), Smad3 (CellSignalingTechnology), nephrin (Invitrogen, CA, USA), SYNPO (Invitrogen), CD19 (CellSignalingTechnology), CD138 (CellSignalingTechnology), SIRT1 (CellSignalingTechnology), and AMPK (CellSignalingTechnology). After primary antibody incubation, the sections were treated with secondary antibodies, Alexa Fluor anti-rabbit 488 or Alexa Fluor anti-mouse 594 (Invitrogen), for 1 h. Nuclear staining was achieved using DAPI (Sigma-Aldrich, MO, USA). Assessment of MPC-5 cell apoptosis was conducted using the Click-It TUNEL kit (Invitrogen), following instructions. Finally, the specimens were visualized using an LSM700 Carl Zeiss confocal microscope (Jena, Germany).
Click it tunel kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Click-iT TUNEL kit is a laboratory tool designed to detect and analyze apoptosis, a form of programmed cell death. The kit utilizes a terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay, which identifies DNA fragmentation, a hallmark of apoptosis. The kit provides a straightforward and sensitive method to visually detect and quantify apoptotic cells.
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Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)
Nephrin, by Thermo Fisher Scientific (1 mentions)
Tgf β1, by Cell Signaling Technology (1 mentions)
Sirt1, by Cell Signaling Technology (1 mentions)
Igg fitc, by Abcam (1 mentions)
Synpo, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 anti rabbit, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 594 anti mouse, by Thermo Fisher Scientific (1 mentions)
Smad3, by Cell Signaling Technology (1 mentions)
Anti ph3 ser10, by Merck Group (1 mentions)
Prolong gold antifade mounting medium, by Thermo Fisher Scientific (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Leica tcs sp5 confocal microscope, by Leica camera (1 mentions)
Dm750, by Leica camera (1 mentions)
Tcs sp5, by Leica camera (1 mentions)
Haematoxylin, by Carl Roth (1 mentions)
Eclipse e400 microscope, by Nikon (1 mentions)
Entellan, by Merck Group (1 mentions)
12 protocols using click it tunel kit
1
Immunofluorescence Analysis of Renal Pathology
2
Histological and Immunostaining Analysis of Testes
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For histological analyses, testes were fixed for 24 h at room temperature (RT) in Bouin’s solution. Tissue was further paraffin-embedded, sectioned (7 μm), and stained with H&E. For immunohistology, testes were essentially fixed in 4% paraformaldehyde for ∼24 h. Paraffin-embedded tissues were further sectioned at 7 μm. For TUNEL and PH3 double immunostaining, sections were deparaffinized followed by antigen retrieval using Sodium Citrate Buffer (10 mM Sodium Citrate, 0.05% Tween 20, pH 6.0). Sections were blocked in PBS containing 5% goat serum for 1 h at RT, followed by TUNEL staining, performed following instructions manufacturer’s instruction (Invitrogen, Click-iT™ TUNEL kit). This was followed by 1° antibody incubation overnight at 4 °C (anti-pH3 (Ser10), 1:100, Millipore) and anti-rabbit AF488 (Invitrogen, 1:1000) 2° antibody incubation for 1 h, RT. Slides were counterstained using DAPI, mounted in Vectashield and imaged as described in below.
3
Apoptotic nuclei quantification via TUNEL
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Cryosections prepared above were also used for TUNEL staining using the Click-iT TUNEL kit (C10619 Invitrogen, ThermoFisher Scientific, Waltham, MA, USA) following the manufacturer’s instructions. And the sections were observed under a confocal microscope (Nikon A1, Nikon, New York, NY, USA). The percentage of apoptotic nuclei was calculated by dividing the total number of TUNEL-stained nuclei by the total number of Hoechst-positive nuclei using ImageJ software (NIH, Bethesda, MD, USA).
4
TUNEL Assay for Apoptotic Cells
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Apoptotic DNA fragments in cells were visualized according to the Click-iT™ TUNEL Kit (C10625, Invitrogen, USA). Cells marked with a brown nucleus were de ned as TUNEL positive cells. The percentage of TUNEL positive cells in each region was estimated after averaging the values of three different regions.
5
Rat Hippocampus Apoptosis Analysis
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Brains of 3 randomly selected rats and / or mice from each experimental group, as described above were used for immunohistological analysis. Histological preparations of frontal sections at the hippocampus level (12 μm) were stained with the Click-IT TUNEL kit (Life Technologies, USA).
6
Hippocampal Neurodegeneration Evaluation
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Brains of 3 randomly selected rats and / or mice from each experimental group, as described above were used for immunohistological analysis. Histological preparations of frontal sections at the hippocampus level (12 μm) were stained with the Click-IT TUNEL kit (Life Technologies, USA).
7
Detecting DNA Fragmentation in PNs
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DNA fragmentation in PNs was detected with Click-iT TUNEL Assays (Invitrogen). The most widely used in situ test for studying DNA fragmentation or strand breaks is the TUNEL assay, which is based on incorporation of modified dUTPs by terminal deoxynucleotidyl transferase at the 3′-OH ends of fragmented DNA. Each Click-iT TUNEL Kit (Thermo Fisher Scientific) contains all the components necessary to detect DNA fragmentation accurately in adherent cells in culture wells.
8
Apoptosis Analysis in Magnetized Pseudo-Islets
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Cell death analysis was performed using pseudo-islets composed of either magnetized β-cells and HUVECs or conventionally cultured β-cells. After culture, the pseudo-islets were fixed with 4% paraformaldehyde (Sigma-Aldrich) overnight at 4°C, embedded in paraffin, and cut into 3 μm thick sections. Terminal deoxynucleotidyl transferase-mediated nick-end labeling (TUNEL) staining was performed using the Click-iT TUNEL kit (C10245; Thermo Fisher Scientific). In brief, sections were deparaffinized in xylene and rehydrated through graded ethanol (100–50%) and vascular endothelial (VE)-water washes, and permeabilized using 0.25% Triton-X for 20 min. Terminal deoxynucleotidyl transferase reaction buffer was applied for 1 h at 60°C before slides were incubated with Click-iT reaction buffer for 30 min. Counterstaining was performed using a 4′,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich) solution with a concentration of 2 μg/mL for 10 min before mounting with ProLong® Gold antifade mounting medium (P36934; Invitrogen™, Darmstadt, Germany).
9
Detecting Apoptosis in Cardiobundles
10
Apoptosis Quantification in Rat Fetal Brain
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The brain of rat fetuses from each experimental group were formalin-fixed, paraffin-embedded, and sectioned at 5-μm thickness sagittal orientation in the brain. Following deparaffinization, we stained apoptotic cells using the Click-iT TUNEL kit (ThermoFisher Inc.) according to manufacturer recommended protocols in stem cell niches and in human brain sections. Briefly, deparaffinized sections were post-fixed and de-permeabilized using Proteinase K. Terminal deoxynucleotidyl transferase (TdT) enzyme and 5-Ethynyl-dUTP (EdUTP) nucleotide solution was added to the sections to incorporate EdUTP into double stranded DNA breaks. Following this, a fluorescent azide was added onto sections and EdUTP was fluorescently detected with Click-iT chemistry. After detection, further immunofluorescence co-staining experiments were conducted to mark Dcx + NPCs, NG2 + OPCs, and nuclei (DAPI). Z-stack images were acquired in the SGZ and SVZ regions using confocal microscopy (Leica SP8). Quantification of TUNEL. 3D images were processed on IMARIS 10.1.1 software (Oxford Instruments). We quantified the volume of TUNEL fluorescence intensity specifically on Dcx + NPCs and NG2 + OPCs along the SGZ and SVZ. Values were averaged between three different sections of the same sample (technical replicates).
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