TUNEL staining was performed using the In Situ Cell Death Detection Kit with TMR red (Roche, Mannheim, Germany) according to the manufacturer’s instructions. The nuclei were counterstained with DAPI Fluoromount-G, and staining was detected via fluorescence microscopy. The ratio of TUNEL-positive cells was calculated as (the number of TUNEL-positive cells/the total number of DAPI-positive cells) × 100%.
In situ cell death detection kit with tmr red
Manufactured by Roche
Sourced in Germany
The In Situ Cell Death Detection Kit with TMR red is a laboratory tool used for the detection and analysis of cell death. It employs the TUNEL (Terminal deoxynucleotidyl transferase dUTP Nick End Labeling) method to label DNA fragmentation, a hallmark of apoptosis or programmed cell death. The kit provides the necessary reagents and protocols to perform this analysis.
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7 protocols using in situ cell death detection kit with tmr red
1
TUNEL Staining for Cell Death
2
Distal Ileum TUNEL Staining
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Sections of the distal ileum were examined. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining was performed using an In Situ Cell Death Detection Kit with TMR red (Roche Diagnostics, Indianapolis, IN) according to the manufacturer’s instructions.
3
Cardiac Myocyte TUNEL Staining Protocol
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TUNEL staining of frozen heart sections and cultured cardiac myocytes was performed using an In Situ Cell Death Detection Kit with TMR red (Roche Applied Science) as described previously25 (link)26 (link). Cryosections (5-μm thick) embedded in O.C.T. compound (Sakura Finetek Japan Co., Ltd) were fixed with 4% paraformaldehyde in PBS and permeabilized with 0.1% Triton X-100. Anti-sarcomeric α-actinin antibody was used for determination of myocytes followed by TUNEL and nuclear staining. DAPI (Sigma-Aldrich Co. LLC) was used for nuclear staining. Three randomly chosen microscopic fields from five different sections in each tissue block were examined for the presence of TUNEL-positive cells.
4
In Situ Cell Death Detection
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Apoptosis in sections was performed using In situ cell death detection kit with TMR Red according to the manufacturer instructions (Roche, West Sussex, UK) (Roche #12 156 792 910) and stored at 4°C until analysis.
5
Blastocyst Fixation and Apoptosis Assay
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Blastocyst fixation (n = 164) and staining was performed in glass plates coated with Sigmacoate siliconizing reagent (Sigma-Aldrich, USA). For fixation, each blastocyst was washed once in 2% BSA/PBS and placed in 4% paraformaldehyde (PFA) for 12 mins at RT. Then the blastocysts were washed twice in 2% BSA/PBS for 5 mins. Subsequently they were placed in a 1% TritonX100 (Sigma-Aldrich, USA) solution for 3 min and washed twice in 2% BSA/PBS for 5 min at RT. Apoptosis was detected using the In-Situ Cell Death Detection Kit with TMR red (Roche, New Jersey, USA) following the manufacturer’s instructions. Specimens were then washed in 2% BSA/PBS and stained with Vectashield DAPI dye (Vector laboratories, USA) for the determination of total cell number. The blastocysts were then placed in individual micro wells of 10-well slides (ThermoFisher, UK), coverslipped and stored in the dark at 4 °C until fluorescence imaging.
6
Apoptosis Evaluation in Cardiomyocytes
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To evaluate apoptosis in cardiomyocytes in heart tissue, terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) staining was performed using an in situ cell death detection kit with TMR red (Roche Molecular Biochemicals, code: 12156792910; Mannheim, Germany), which was used to label the apoptotic cells. In brief, 4 µm-thick formalin-fixed, paraffin-embedded heart sections were deparaffinized and dehydrated as described above and boiled for 2 minutes in 0.1 M citrate buffer, pH = 6, using a microwave. After washing with PBS, the sections were covered with TUNEL reaction mixture for 60 minutes at 37 °C in a humidified atmosphere in the dark. After three washes with PBS, the sections were evaluated under a fluorescence microscope. Positive and negative controls were generated according to the manufacturer’s instructions.
7
Quantifying Chondrocyte Cell Death
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We measured cell death using the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining method and the In Situ Cell Death Detection Kit with TMR red (Roche, Germany). Following the manufacturer's guidelines, cells were fixed with 4% paraformaldehyde in PBS and were permeabilized by incubation in a permeabilization solution (0.1% Triton X-100 in 0.1% sodium citrate) for 2 min on ice. The TUNEL reaction mixture containing terminal deoxynucleotidyl transferase and rhodamine (the labeling dye) was added to the slides and incubated at 37 °C in a humidified chamber in the dark for 60 min. The reaction was stopped by a blocking buffer (0.1% Triton X-100/0.5% BSA in PBS). The cells nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI), and only dead cells were stained red by rhodamine. The slides were observed under a fluorescence microscope (Nikon TE300) with an excitation wavelength of 580 nm for rhodamine and 365 nm for DAPI. Stained cells were counted in 5 microscopic fields on each slide. Data were analyzed using Image-Pro Plus analytical software (Media Cybernetics, Silver Spring, MD). The death rate of chondrocytes was defined as the ratio of red-stained cells (dead cells) to blue-stained cells (total cells).
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