Tunel apoptosis Detection Kit



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TUNEL Apoptosis Detection Kit Cat. No. L00299

(For Adherent Cells, FITC-labeled POD )

Technical Manual No. 0265 Version 01132011








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Description ………………………………………………………………. ……………….

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II

Key Features ……………………………………………………………. . ………………..

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III

Kit Contents ………………………………………………………. . ………………………

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IV

Storage..………………………………………………………………………………………

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V

Protocol ………………………………………………. ……………………………………

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VI

Related Products…………………………………………………………………………..

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VII

Troubleshooting …….………………………………………………………………. …….

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VIII

Ordering Information….…………………………………………………………….……

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I. DESCRIPTION

The TUNEL Apoptosis Detection Kit for Adherent Cells (FITC-labeled POD) (Cat. No. L00299) is one of GenScript’s newly introduced products. The kit can detect fragmented DNA in the nucleus during apoptosis. In this modified TUNEL assay kit, fluorescein-labeled nucleotides bind with the DNA 3'-OH ends using natural or recombinant terminal deoxynucleotidyl transferase (TdT or rTdT). The fluorescence could be observed by fluorescence microscope. And then the compound of anti-fluorescein antibody and HRP is bound to these fluorescein labeled nucleotides, which are detected using the peroxidase substrate, hydrogen peroxide, and 3,3’-diaminobenzidine (DAB), a stable chromogen. Using this procedure, apoptotic nuclei are stained dark brown.




  1. KEY FEACTURE

      • Simplified Procedure: The kit contains ready-to-use reagents, including DAB and DNase I.

      • Enhanced Sensitivity: This kit can assay the cells during the early stages of apoptosis.

      • Enhanced Specificity: The kit can stain apoptotic cells.

      • Streamlined Process: The entire procedure takes about three hours.

      • Increased Convenience: The results can be observed by fluorescence microscope and light

microscope.

  1. KIT CONTENTS

The TUNEL Apoptosis Detection Kit is available. L00299 is for detection using fluorescein Labeled nucleotides (FITC-12-dUTP), HRP-labeled anti-FITC antibody, TdT and DAB.

Components

Cat. No. L00299

20 Assays

Cat. No. L00299

50 Assays

Cat. No. L00299

100 Assays

Storage Conditions

Equilibration Buffer

1.0 ml

2.5ml

5.0 ml

-20°C

FITC-12-dUTP

20 µl

50 µl

100 µl

-20°C

TdT

80 µl

200 µl

400 µl

-20°C

HRP-labeled Anti-FITC Antibody

200 µl

500 µl

1000 µl

-20°C

DAB

2 mg

5 mg

10 mg

-20°C

DNase I

0.2 ml

0.5 ml

1 ml

-20°C

1×DNase I buffer

0.2 ml

0.5 ml

1 ml

4°C
The concentration of DNase I is 50 U/µl.

IV. STORAGE

Store the kit at -20°C. It will remain stable for one year.


V. TUNEL Apoptosis Detection Kit PROTOCOL

1Before use, prepare the following:

Fixation Solution: 4% paraformaldehyde in PBS, pH 7.4, freshly prepared.

Blocking Solution: 3% H2O2 in methanol. e.g. 1ml 30% H2O2 + 9ml methanol.

Permeabilization Solution: 0.1% Triton X-100 and 0.1% sodium citrate in water, freshly prepared.


Note:

1. Please centrifuge the reagents in the kit before use.

2. Please prepare the proper amount of TUNEL Reaction Mixture according to the amount of the samples to save reagent.

3. The DAB is powder, please dissolve the DAB powder in PBS to make 20×DAB buffer (10 mg/ml DBA buffer) before use.



Adherent Cells, Cell Smears, and Cytospin Preparations



Controls:

Negative control: Employ the cells or sections as described the labeling protocol. Label solution but do not add any Terminal Deoxynucleotidyl Transferase (TdT) to the TUNEL Reaction Mixture.

Positive control: Before beginning the labeling procedures, incubate the fixed and permeabilized cells or sections with 100 μl DNase I Solution for 10 minutes at 15-25°C to induce DNA strand degradation.

(DNase I Solution contains 10000 U/ml-20000U/ml DNase I (grade I) depending on the sample to be stained in 1X DNase I buffer. One example of 1X DNase I buffer is 10 mM CaCl2, 6 mM MgCl2, and 10 mM NaCl in 40 mM Tris-HCl, pH 7.9)



2. Labeling Protocol


Rinse slides with PBS two times for five minutes each time and then keep the area around the samples dry.







Add 50 µl TUNEL Reaction Mixture to samples. Add a coverslip and incubate for 60 minutes at 37°C under wet conditions, protected from light.

(TUNEL Reaction Mixture contains 45 µl Equilibration Buffer, 1 µl FITC-12-dUTP and

4 µl TdT, freshly prepared.)

Note: Add 50 µl Label Solution to the negative control. To ensure a homogeneous dispersal of TUNEL Reaction Mixture across the cell monolayer and to avoid loss to evaporation, the samples should be covered with parafilm or a coverslip during incubation.




Rinse slides with PBS three times for five minutes each time.





Assay with a fluorescence microscope using excitation wave 450-500 nm and emission wave 515-565 nm (green).



Keep the area around the samples dry with filter paper.






Add 50 µl Anti-fluorescein Antibody Solution on samples. Incubate slide under wet conditions for 30 minutes at 37°C.

(The Anti-fluorescein antibody solution contains10 µl anti-fluorescein antibody

in 40 µl PBS.)

Note: To ensure the homogeneous dispersal of Anti-fluorescein Antibody Solution across

thecell monolayer and to avoid loss to evaporation, the samples should be covered with

parafilm or a coverslip during incubation.








Rinse slide three times with PBS for five minutes each time.

Add 50 µl Streptavidin-HRP Solution to samples. Incubate the slide under wet conditions, protected from light, for 30 minutes at 37°C.

[Same font](Streptavidin-HRP solution contains 0.5µL Streptavidin-HRP in 99.5µL PBS buffer.)[Same font]

Note: To ensure the homogeneous dispersal of Streptavidin-HRP Solution across the cell monolayer and to avoid loss to evaporation, the samples should be covered with parafilm or a coverslip during incubation.









Add 50-100 µl DAB Substrate and incubate slide for 10 minutes at 15-25°C.

(The DAB Substrate contains 5 µl 20×DAB buffer* and 1 µl 30% H2O2 in 94 µl PBS buffer, freshly prepared.)









Rinse slides three times with PBS. Mount under a glass coverslip (such as with PBS/glycerol) and analyze with light microscope.

(Alternative: Samples can be counterstained prior to analysis by light microscope.)



20×DAB buffer (1 mg/ml DAB buffer) contains 10 mg DAB dissolved in 1.0 ml PBS.
*20×DAB buffer (10 mg/ml DAB buffer) contains 10 mg DAB dissolved in 1.0 ml PBS.


VI. RELATED PRODUCTS

TUNEL Universal Apoptosis Detection Kit (Biotin labeled POD), Cat. No. L00290

TUNEL Apoptosis Detection Kit for Adherent Cells (Biotin labeled POD), Cat. No. L00296

TUNEL Apoptosis Detection Kit for Paraffin-embedded Tissue Sections (Biotin labeled POD), Cat. No. L00297

TUNEL Apoptosis Detection Kit for Paraffin-embedded Tissue Sections (FITC labeled POD), Cat. No. L00300

TUNEL Apoptosis Detection Kit for Cryopreserved Tissue Sections (FITC labeled POD), Cat. No. L00301


VII. TROUBLESHOOTING

TdT dilution buffer* contains 150 mM KCl, 1 mM 2-mercaptoethanol, and 50% glycerol in 60 mM KPB, pH 7.2.

Problem

Step/Reagent

Possible cause

Solution

High background




Fixation

Formalin fixation leads to a yellowish stain in cells containing melanin precursors.

Use methanol for fixation. However, this may lead to reduced sensitivity.

TUNEL reaction

The concentration of the labeling mix is too high.

Reduce concentration of labeling mix from 10% to 50%.

Converter solution

There is endogenous peroxidase activity.

Prior to cell permeabilization, block endogenous peroxidase by incubating for 10 minutes in methanol containing 3% H2O2 at 15-25°C.

Streptavidin-HRP has engaged in non-specific binding.

• Block with anti-mouse serum.

• Block with PBS containing 3% BSA for 20 minutes.

• Reduce the concentration of

Streptavidin-HRP Solution to 50%.



The DAB incubation time is too long.

Reduce the time of incubation.



Sample

Mycoplasma contamination


Use a mycoplasma detection kit.


Highly proliferating

cells


Double staining with Annexin-V-Fluos or a similar substance.

Note: High background may make measuring with microplate readers impractical.




Fixation


After fixation, nuclease activity is still high.

Block with the buffer containing dUTP and dATP

Non-specific staining




TUNEL reaction

The concentration of TdT is too high.

Reduce concentration of TdT from 10% to 50% with TdT dilution buffer*.

Low rate of labeling



Fixation

Ethanol and methanol can lead to diminished labeling (chromatins are not cross-linked with proteins during fixation; they are lost during the procedure steps).

Fixate using 4% paraformaldehyde buffer, formalin, or glutaraldehyde.

Extensive fixation leads to excessive cross-linkage with proteins.

Reduce fixation time or fix by using 2% paraformaldehyde PBS buffer (pH 7.4).

Permeabilization


The permeabilization step is too short and the reagents can’t reach their target molecules.

• Increase the incubation time.

• Incubate at a higher temperature (such as 15-25°C).

• Optimize the concentration and action time of proteinase K. (e.g. 400ug/ml for 5 minutes)

• Incubate with 0.1 M sodium citrate at 70°C for 30 minutes.


No signal in positive

control


DNase treatment

The concentration of DNase I buffer is too low.

• Incubate with 30000 U/ml DNase I Solution or higher for 30 minutes at 37°C and then rinse with PBS.

Weak signals





Counterstaining


The dye is not suitable.

Counterstain with 5% methyl green in 0.1 M veronal acetate, pH 4.0 or Hematoxylin.




VIII. ORDERING INFORMATION

TUNEL Apoptosis Detection Kit for Adherent Cells (FITC labeled POD), Cat. No. L00299

GenScript USA Inc.

860 Centennial Ave., Piscataway, NJ 08854

Tel: 1-877-436-7472

Fax: 1-732-210-0262

E-mail: product@genscriptt.com

Web: www.genscriptt.com



For Research Use Only.





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