During apoptosis, chromosome breakage is a gradual and phased process, with chromosomes first degraded to large fragments of 50-300 kb by endogenous nucleic acid hydrolases. Then, about 30% of the chromosomes are randomly cut off in nucleosomes under the action of endonucleases to form nucleosome-DNA multimers of 180-200 bp. Genomic DNA fragmentation results in a large number of exposures3'-OH-terminus, which labels deoxyribonucleotides and derivatives formed by fluorescein (FITC), peroxidase, alkaline phosphorylase, or biotin to DNA 3 in response to terminal deoxynucleotidyl transferase (TDT).'- ends, which allows for the detection of apoptotic cells. This method is known as deoxyribonucleotide terminal transferase-mediated nick end labeling (TUNEL). Since normal or proliferating cells have little to no DNA breakage, there is none3'-OH is formed and is rarely able to be stained. Therefore, apoptosis can be detected by fluorescence microscopy or flow cytometry.
Fig. Schematic diagram of TUNEL staining.
Customer** places an order - order Confirmation of experimental materials - sample preparation (optional) - sections (paraffin sections and frozen sections only) - TUNEL staining (optional fluorescent labeling or biotin labeling) - optional fluorescence microscope photography or follow-up DAB staining - apoptosis index analysis - issuance of experimental report - result delivery.
Applicable scenarios
1.Detect the effect of external stimuli on plant cells;
2.Detect changes in plant cells at different stages.
Experimental cycle
Experimental cycle: about 14-21 working days, the specific time can be called for details.
Sample Acceptance Criteria
1. Paraffin sections: transported at room temperature.
Root tip, flower bud and other parts: 50% FAA fixation.
Branches, leaves, fruits, etc.: 70% FAA
2. Ice cutting**: no need to fix, -20 preservation, dry ice + a small amount of ice pack transportation (communicate in advance).
Advantages of TUNEL staining
TUNEL is currently the most sensitive, rapid and specific method for the in-situ detection of apoptosis, which has been widely used in the field of biological and medical research, which can detect the generation of DN** segment in the early stage of apoptosis, and can be directly used on tissue sections to observe the location of apoptosis, so it is an apoptosis analysis method that has been widely accepted and used so farIt can be used in a wide range of applications, including all types of adherent cells (typically cell crawlers), suspension cells (or cell suspensions), and tissue sections.
Disadvantages of TUNEL staining
1. Necrotic cells also have DNA lob point formation, and can also show positive TUNEL reaction, so the specificity is poor;
2. When TUNEL is combined with immunohistochemistry (IHC) detection, the fixation process has a great impact on the detection, and the size and thickness of the slice will directly affect the fixation effect, resulting in differences in results.
Customer orders and fill in project information
To register or log in to our official website, please fill in the project name, select the project type, fill in the personal information and the specific information required to submit the project according to the page prompts, including:
1. Sample type and sample size;
2. Final detection method;
3. Abundant relevant materials and literature as much as possible.
Experiment information
During the experiment, the customer can log in to the management system at any time to view the real-time progress of the project. When the experiment is completed, the system will automatically notify the customer via SMS and send the experiment report to view**. After the experiment is completed, the experimental report and test results can be viewed or printed, and permanently retained.
Conclusion criteria
1. The experimental process is correct: no cracks, wrinkles and uneven thickness.
2. It can be optimized once for free: re-sectioning and replacing the staining.
Deliverables
1. Raw data of all experiments (including experimental procedures and result analysis reports, etc.);
2. TUNEL staining results (original detection**, etc.);
3. Embedding and staining pieces (limited to customers providing tissues).
Fig. TUNEL staining was used to detect programmed death of crown cells in Cactus cactus root (AL) TuneL-positive nuclei with fluorescent green fluorescence were labeled using TUNEL reaction FITCNuclei stained with DAPI fluoresce blue. (A) FITC and (B) DAPI, PCD positive control: apex of the first stage salt-treated root. (c) FETC, (d) DAPI, nuclear fragmentation in root hair cells. (E) FITC, (F) DAPI, TUNEL-positive nuclei in root hair cells. (G) FITC, (H) DAPI, TUNEL-positive nuclei in root hair cells. (I) FITC, (J) DAPI, Segmentation of DN** in root hair cells near the vertex after meristem depletion and growth cessation. (K) FITC, (L) DAPI, single tunel-positive nuclei in root caps. (m) Contrast image of the same part as k and l. Open meristem and root cap are shown. Scale bar: (a m) 50 m.
References
shishkova s, dubrovsky jg. developmental programmed cell death in primary roots of sonoran desert cactaceae. am j bot. 2005;92(9):1590-1594.