For TUNEL staining the Click‐iT TUNEL Alexa Fluor 647 Kit (Invitrogen, Carlsbad, USA) was used. Fish were euthanized in ice water, followed by fixation of the brain inside the skull in 4% PFA at 4°C. Subsequently, the brains were carefully removed from the skulls and incubated in 11 µg/ml Proteinase K in PBST (PBS containing 0.2% Triton X‐100) at room temperature for 40 min. Then the brains were incubated in fixative again (4% PFA) at room temperature for 20 min. After washing with PBST the brains were incubated in the reaction buffer at room temperature for 30 min, followed by overnight incubation in the reaction cocktail at room temperature, according to the manufacturer's instructions. After washing with 3% w/v BSA in PBST the brains were incubated in the Click‐iT reaction cocktail at room temperature for 3 h, followed by washing with 3% BSA in PBST. Then the brains were incubated in PBST containing 1% v/v DMSO and 1% BSA at room temperature for 2 h. The brains were incubated with primary antibody (l ‐plastin, gift from Yi Feng, University of Edinburgh, 1:500) in 5% BSA in PBST at 4°C for 72 h. Subsequently, brains were incubated overnight at 4°C with fluorescently labeled secondary antibody (DyLight Alexa 488 1:250) and Hoechst in PBST containing 2% BSA. Brains were sections as described and sections were mounted in Vectashield mounting medium.
Click it tunel alexa fluor 647 kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Click-iT TUNEL Alexa Fluor 647 Kit is a molecular biology tool used to detect and quantify apoptosis, a form of programmed cell death. The kit utilizes terminal deoxynucleotidyl transferase (TdT) to incorporate Alexa Fluor 647-labeled nucleotides into the free 3'-hydroxyl ends of DNA fragments, which are generated during apoptosis. This allows for the visualization and analysis of apoptotic cells using fluorescence microscopy or flow cytometry.
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5 protocols using click it tunel alexa fluor 647 kit
1
TUNEL Assay for Apoptosis Detection in Fish Brains
2
Detecting Beta-Cell Death in Zebrafish
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To detect beta-cell death we used the Click-iT® TUNEL Alexa Fluor 647 kit (Invitrogen: C10247). Larvae were fixed in 4% PFA in PBS for 48 hours at 4 degrees under constant mixing. The larvae were washed in 1XPBS and dissected to remove the skin covering the islet. Dissected larvae were permeabilized by incubation in 1% Triton X-100 in 1X PBS (1%PBST) for 20 minutes (3 times). Next, we prepared the TdT reaction according to kit protocol. 200 µl of TdT reaction mixture were used per 6 larvae. Larvae were incubated in the TdT reaction at 37 °C for one hour. The larvae were washed 2 times each for 2 minutes in 4% BSA in 1%PBST. The Click it reaction was prepared according to Kit protocol and 200 µl of reaction mixture were used per 6 larvae. Larvae were incubated at room temperature and protected from light for 30 minutes. The larvae were washed with 4% BSA in 1%PBST for 5 minutes. Finally, to detect the ins:CFP signal, which is lost following the Click-iT®reaction, we performed antibody staining using anti-GFP antibody as described in the immunostaining and imaging section.
3
TUNEL Staining of Larval Brains
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For staining larvae were euthanized on ice, followed by fixation in 4% paraformaldehyde in PBS at 4°C overnight, permeabilized by proteinase K (10 μg/mL) in PBST treatment for 40 min and refixed in PFA for 20 min at room temperature. TUNEL staining was performed as previously described using the Click‐iT TUNEL Alexa Fluor 647 Kit (Invitrogen, Carlsbad) (C10247) (van Ham, Mapes, Kokel, & Peterson, 2010 ). TUNEL+ cells in the whole brain were quantified.
4
Neuronal Apoptosis Assessment After TBI
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At the end of the beam walking test (34 days after TBI), the rats were anesthetized with Zoletil-100 (50 mg/kg, intraperitoneal), perfused with 4% paraformaldehyde, and then decapitated. The brain was extracted and examined by confocal microscopy. Brains from all animals used for immunohistochemical assays were fixed in 4% paraformaldehyde and cryoprotected in 20% sucrose before storage in isopentane at −70 °C. Coronal sections (15 μm) were prepared for morphological and immunohistochemical assay with a Leica CM1510S-1 cryostat (Leica Microsystems, Wetzlar, Germany). The frontal slices were collected at the level of the hippocampus (from bregma −3 mm to −4.3 mm) according to the atlas of Paxinos and Watson [33 ]. Six alternate series of sections were mounted on SuperFrost Plus slides (Menzel GmbH, Berlin, Germany).
For confocal microscopy, sections were preincubated in blocking solution (2% bovine serum albumin diluted in PBS with 0.1% Tween-20) for 1 h at room temperature, and then processed with Click-iT TUNEL Alexa Fluor 647 kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s protocol. After rinsing in PBS, the sections were subsequently incubated with 4′,6-diamidino-2-phenylindole (DAPI) for 10 min (1:10,000; Sigma-Aldrich, St. Louis, MO, USA). Fluorescent images were captured by Olympus confocal system FV3000 (Olympus, Tokyo, Japan).
For confocal microscopy, sections were preincubated in blocking solution (2% bovine serum albumin diluted in PBS with 0.1% Tween-20) for 1 h at room temperature, and then processed with Click-iT TUNEL Alexa Fluor 647 kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s protocol. After rinsing in PBS, the sections were subsequently incubated with 4′,6-diamidino-2-phenylindole (DAPI) for 10 min (1:10,000; Sigma-Aldrich, St. Louis, MO, USA). Fluorescent images were captured by Olympus confocal system FV3000 (Olympus, Tokyo, Japan).
5
TUNEL Assay for Apoptosis Detection
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The TUNEL assay was carried out using Click-iT TUNEL Alexa Fluor 647 kit (Thermo Fisher Scientific, Middletown, VA, USA), according to the manufacturer’s instructions. Briefly, HeLa cells at a concentration of 1.5 × 105 cells/mL were cultured in glass-bottom confocal culture dishes, and incubated under 5% CO2 for 24 h at 37 °C. The cells were rinsed with PBS and treated with (0, 1.0, and 10) µM of β-cryptoxanthin for 24 h, and then incubated with 4% (v/v) formaldehyde for 20 min, followed by 0.25% (v/v) Triton X-100 for 25 min. TUNEL images were obtained by Olympus FLUOVIEW FV1200 microscope (Olympus Corporation, Japan). The TUNEL-positive cells were assessed by manual counting of at least 300 cells. Cells with only nuclear staining were considered TUNEL-positive (cytoplasmic staining was not considered).
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