After intracardiac paraformaldehyde perfusion, approximately 2 mm brain tissues of rats were obtained from two points, anterior and posterior to the optic chiasm. Brain specimens were immersed in 4% paraformaldehyde individually, fixatived for 24 h and embedded in paraffin for the slicing of 3-μm sections. TUNEL analysis was performed according to the instructions of the TUNEL-POD kit (Roche Applied Science, Germany). Positively labeled nuclei (brown color) were identified from unstained nuclei (blue color). Four visual fields were randomly seleted under a high magnification microscope (100×) were chosen in the frontoparietal cortex of the ischaemic hemisphere. The apoptotic index was calculated based on the percentage of positive apoptotic cells.
Tunel pod kit
Manufactured by Roche
Sourced in Germany, United States
The TUNEL-POD kit is a laboratory tool designed for the analysis of apoptosis, a type of programmed cell death. The kit provides a method for the detection and quantification of DNA fragmentation, a hallmark of apoptosis. The core function of the TUNEL-POD kit is to enable researchers to visualize and measure the extent of apoptosis in their samples.
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7 protocols using tunel pod kit
1
Apoptosis Quantification in Ischemic Rat Brain
2
TUNEL Staining for Apoptosis Quantification
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The TUNEL staining could examine the 3’-OH terminals of damaged DNA in late apoptosis cells. Therefore, the TUNEL was used to observe apoptosis level in the myocardial infarct size of rats. The TUNEL assay was performed by utilizing the TUNEL-POD kit (Roche, Germany), as previously described [56 (link)]. Briefly, the isolated myocardial tissues were embedded with paraffin and sectioned into slices soaked and washed by xylene and hydrated in gradient alcohol. Then the paraffin section of myocardial tissues was treated by proteinase K working solution for 30 min, with PBS washing for three times (3 min/wash). The 3% H2O2 solution was used to inactivate the endogenous peroxidase in the myocardial tissues for 10 min, with three times (3 min/wash) of PBS rinse. The paraffin section was treated with TUNEL reaction mixture (biotin dUTP and terminal deoxynucleotidyl transferase (TdT)) in a dark box at 37° C for 1 h, washed by PBS three times. An equal volume of solution with biotin dUTP to replace the TUNEL reaction mixture was used as the negative control. Samples were analyzed under fluorescence microscope (Olympus, Japan) using an excitation wavelength in the range of 450 nm and detection in the range of 565 nm.
3
Immunohistochemistry Analysis of Lung Cancer
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Standard-protocol IHC was done on Human Lung Cancer Survey tissue microarrays (US Biomax, Milan, Italy) and formalin-fixed paraffin-embedded (FFPE) XT tissues. The UltraTek HRP Anti-Polyvalent (DAB) Staining System (ScyTek, Logan, UT, USA) was used for blocking and detection; apoptosis was detected with TUNEL-POD kit (Roche, Monza, Italy). Labeling was detected with the UltraTek HRP Anti-Polyvalent (DAB) Staining System (ScyTek). Stained sections were examined with a Leica DM1000 microscope (Leica Microsystems Srl, Milan, Italy), photographed with a Jenoptik ProgRes SpeedXT Core 3 camera (Jenoptik AG, Vienna, Austria), and analyzed with ProgRes Capture Pro 2.8 software (Jenoptik AG).
Protein expression was evaluated with histoscore method (H-score): staining intensity was rated from 0 (negative) to 3 (strong) for each sample by two independent observers and the mean was used. H-score was calculated multiplying the number of cells within each category in at least three different × 200 fields. Human protein KI-67 expression and TUNEL scores were reported as percentages of labeled cells with respect to total cells.
Protein expression was evaluated with histoscore method (H-score): staining intensity was rated from 0 (negative) to 3 (strong) for each sample by two independent observers and the mean was used. H-score was calculated multiplying the number of cells within each category in at least three different × 200 fields. Human protein KI-67 expression and TUNEL scores were reported as percentages of labeled cells with respect to total cells.
4
TUNEL Assay for Apoptosis Detection
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To detect cells undergoing apoptosis, TUNEL technique was performed according to the manufacturer's protocol supplied within the TUNEL-pod kit (Roche, USA). At first, the brain sections of hippocampus CA1 region were immersed in 3% H2O2 for inactivation of endogenous hydrogen peroxidase activity. After rinsing with PBS, the sections were incubated with proteinase K solution at 37°C for 20 min to enhance the permeability. Then, the sections were incubated for 60 min with TUNEL reaction mixture and for 30 min with converter-POD at 37°C. At last, after incubated for 10 min with DAB substrate solution (Zymed, USA), the sections were counterstained with 0.5% methyl green. Positive and negative controls were carried out on slides from the same block. Stained slides were randomly observed under a high magnification microscope and the pathological changes near the injection needle were photographed. The image pictures were processed by the America IPP6.0 software and the positive cells (brown) were calculated.
5
Quantifying Apoptosis in Hearing Cells
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TUNEL assay was used to quantitatively determine the apoptotic cells pretreated with A666-DEX-NP. Briefly, HEI-OC1 cells were seeded on glass cover slips in 24-well culture plates containing growth medium. After the monolayers achieved 80% confluence, the medium was replaced with DEX, DEX-NP, or A666-DEX-NP at the same DEX concentration of 80 ng/mL. After 4 hours, the culture medium was replaced with medium containing 30 µM CDDP. The medium with saline alone was used as the control. After 24 h incubation, the cells were washed with cold PBS, fixed in 4% PFA. Next, the cover glasses were washed in PBS and incubated with the TUNEL reaction mixture for 1 h at 37°C. After washing, the slides were incubated with Hoechst 33342 for 30 min at room temperature. Apoptotic cells were detected in situ utilizing the TUNEL assay (TUNEL POD kit, Roche, Germany). The stained cells were observed under LSM-510 CLSM using an excitation wavelength in the range of 450-550 nm and a detection wavelength in the range of 515-565 nm. TUNEL positive cells were counted under a 200× magnification field in triplicate.
6
TUNEL Assay for Apoptosis Detection
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The TdT-mediated dUTP-biotin nick end labeling (TUNEL) assay was performed using a TUNEL-POD kit according to the manufacturer's recommendations (Roche, Germany). Briefly, the sections were dewaxed and rehydrated according to standard protocols (by heating at 60°C, followed by washing in xylene and rehydration through a graded series of ethanol and double distilled water). The sections were incubated with 15 g/ml proteinase K for 15 min at room temperature and then washed in PBS. Then, the sections were immersed in buffer containing terminal deoxynucleotidyl transferase and incubated for 90 min at 37°C in a humid atmosphere. After the sections were washed again in PBS, they were incubated with anti-digoxigenin conjugate for 30 min at room temperature. After the sections were washed in PBS and the color of the peroxidase substrate was developed with diaminobenzidine, the signals were examined by microscopy. Labeled nuclei were counted in 10 independent, randomly selected fields.
7
TUNEL Assay for Apoptosis Quantification
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TUNEL technique was performed according to the manufacturer's protocol supplied within the TUNEL pod kit (Roche, USA). The dewaxed slices were placed into 3% hydrogen peroxide in methanol for 15 min and were added 100 μl protease K (20 μg/ml) for 20 min. After washing with PBS, TUNEL reaction solution was added to the slices at 37°C for 1 h, and then, the POD was added to the slices. Finally, the slices were rinsed 3 times and stained with DAB and hematoxylin. The number of viable TUNEL-positive neurons in the hippocampus was observed by an Olympus fluorescence positive microscope.
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