In Situ Cell Death Detection Kit, POD
Kit for immunohistochemical detection and quantification of apop-tosis (programmed cell death) at single cell level, based on labeling of DNA strand breaks (TUNEL technology): Analysis by light microscopy.
Cat. No. 1 684 8171 Kit (50 tests)
Store at Ϫ15 to Ϫ25°C
Instruction Manual
Version 3, January 2003
1. Preface
1.1
Table of contents
1. Preface ......................................................................................................................21.1Table of contents ............................................................................................................................21.2 Kit contents .......................................................................................................................................32. Introduction ..............................................................................................................52.1Product overview ............................................................................................................................52.2 Background information ..............................................................................................................83. Procedures and required materials .................................................................103.1Flow chart ........................................................................................................................................103.2Preparation of sample material ...............................................................................................113.2.1Adherent cells, cell smears and cytospin preparations .................................................113.2.2 Tissue sections ..............................................................................................................................123.2.2.1 Treatment of paraffin-embedded tissue ..............................................................................123.2.2.2Treatment of cryopreserved tissue ........................................................................................143.3Labeling protocol ..........................................................................................................................153.3.1 Before you begin ..........................................................................................................................153.3.2Labeling protocol for adherent cells, cell smears, cytospin preparations,
and tissues.......................................................................................................................................16
3.3.3 Labeling protocol for difficult tissue .....................................................................................173.4 Signal conversion .........................................................................................................................184. Appendix ................................................................................................................194.1 Trouble-shooting ..........................................................................................................................194.2 References ......................................................................................................................................224.3 Related products ..........................................................................................................................23
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1.2 Kit contentsCaution
Kit contents
The Label solution contains cacodylate, toxic by inhalation and swal-lowed, and cobalt dichloride, which may cause cancer by inhalation.
Avoid exposure and obtain special instructions before use.
When using do not eat, drink or smoke. After contact with skin, wash immediately with plenty of water. In case of accident or if you feel unwell seek medical advice immediately (show label where possible).Collect the supernatants from the labeling reactions in a tightly closed, non-breakable container and indicate contents. Discard as regulated for toxic waste.
Please refer to the following table for the contents of the kit.Vial/Label
Contents
Cap1Enzyme Solution•Terminal deoxynucleotidyl transferase
blue
from calf thymus (EC 2.7.7.31), recom-binant in E. coli, in storage buffer•10× conc.•5 × 50 l2
Label Solution
•Nucleotide mixture in reaction bufferviolet•1× conc.•5 × 550 l
3Converter-POD
•Anti-fluorescein antibody, Fab frag-yellow
ment from sheep, conjugated with horse-radish peroxidase (POD)•Ready-to-use•3.5 ml
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1.2 Kit contents, continuedAdditional equipment required
Procedure
•Adherent cells, cell smears and cytospin preparations (section 3.2.1)
•Cryopreserved tissue (section 3.2.2.2)
In addition to the reagents listed above, you have to prepare several solutions. In the table you will find an overview about the equipment which is needed for the different procedures.
Detailed information is given in front of each procedure.
Equipment
Reagents
•Washing buffer: Phosphate buffered saline (PBS*)
•Blocking solution: 3% H2O2 in methanol•Fixation solution: 4% Paraformaldehyde in PBS, pH 7.4, freshly prepared
•Permeabilisation solution: 0.1% Triton X-100 in 0.1% sodium citrate, freshly pre-pared (6)•Xylene and ethanol (absolute, 95%, 90%, 80%, 70%, diluted in double distilled water)•Washing buffer: PBS*
•Proteinase K*, nuclease, working solution: [10-20 µg/ml in 10 mM Tris/HCl, pH 7.4-8]Alternative treatments•Permeabilisation solution: (0.1% Triton1) X–100, 0.1% sodium citrate) , freshly prepared•Pepsin* (0.25% - 0.5% in HCl, pH 2) or trypsin*, 0.01 N HCl, nuclease free
•0.1 M Citrate buffer, pH 6 for microwave irradiation•Micrococcal nuclease or •DNase I, grade I *
•Parafilm or Washing buffer: PBS*coverslips•Humidified chamber
•Plastic jar•Citrate buffer, 0.1 M, pH 6.0.•Microwave•Washing buffer: PBS*
•Humidified •Tris-HCl, 0.1 M pH 7.5, containing 3% BSA* chamberand 20% normal bovine serum•Humidified •Washing buffer: PBS*chamber•DAB Metal Enhanced Substrate Set* or •Parafilm or alternative POD substrates coverslip•Mounting medium for light microscopy
Preparation of sample material (section 3.2)
Paraffin-embedded tissue
(section 3.2.2.1)
Labeling protocol (section 3.3)Positive control (section 3.3.1)
Adherent cells, cellsmears, cytospin
preparations, and tissues (section 3.3.2) Difficult tissue (section 3.3.3)
Signal conversion (section 3.4)
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2. Introduction
2.1
Product overview
Test principle
Application
Cleavage of genomic DNA during apoptosis may yield double-stranded, low molecular weight DNA fragments (mono- and oligonu-cleosomes) as well as single strand breaks (“nicks”) in high molecular weight DNA.
Those DNA strand breaks can be identified by labeling free 3’-OH ter-mini with modified nucleotides in an enzymatic reaction.
StageDescription1Labeling of DNA strand breaks, by Terminal deoxynucleotidyl transferase (TdT), which catalyzes polymerization of labeled nucleotides to free 3’-OH DNA ends in a template-indepen-dent manner (TUNEL-reaction).2Incorporated fluorescein is detected by anti-fluorescein anti-body Fab fragments from sheep, conjugated with horse-radish peroxidase (POD). After substrate reaction, stained cells can be analyzed under light microscope.3After substrate reaction, stained cells can be analyzed under light microscope.fluorefluorescein-labeledFixed cells withscein-labeledantibody conjugatedAnti-fluoresceinAnti-fluoresceinSubstrate for POSubstrate for PODDNA strand breakswith POwith POD Fig. 1: Test principle
The In Situ Cell Death Detection Kit is designed as a precise, fast and
simple, non-radioactive technique to detect and quantify apoptotic cell death at single cell level in cells and tissues. Thus, the In Situ Cell Death Detection Kit can be used in many different assay systems. Examples are:
•Detection of individual apoptotic cells in frozen and formalin fixed tissue sections in basic research and routine pathology.
•Determination of sensitivity of malignant cells to drug induced apo-ptosis in cancer research and clinical oncology.
•Typing of cells undergoing cell death in heterogeneous populations by double staining procedures (6).
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2.1Product overview, continued
The TUNEL reaction preferentially labels DNA strand breaks gener-ated during apoptosis. This allows discrimination of apoptosis from necrosis and from primary DNA strand breaks induced by cytostatic drugs or irradiation (3, 4).
False negative results: DNA cleavage can be absent or incomplete in Specificity
Test interference
Sample material
Assay timeNumber of testsKit storage/stability
some forms of apoptotic cell death (37). Sterical hindrance such as extracellular matrix components can prevent access of TdT to DNA strand breaks. In either case false negative results can be obtained.False positive results: Extensive DNA fragmentation may occur in late stages of necrosis (4, 38).
DNA strand breaks may also be prominent in cell populations with high proliferative or metabolic activity. In either case false positive results may be obtained. To confirm apoptotic mode of cell death, the morphology of respective cells should be examined very carefully. Morphological changes during apoptosis have a characteristic pattern. Therefore evaluation of cell morphology is an important parameter in situations where there is any ambiguity regarding interpretation of results.
•Cytospin and cell smear preparations
•Adherent cells cultured on chamber slides (31)
•Frozen or formalin-fixed, paraffin-embedded tissue sections (1, 25, 26, 29, 30, 32–34, 36, 39)2–3 hours, excluding culture, fixation and permeabilisation of cells and preparation of tissue sections.The kit is designed for 50 tests.
The unopened kit is stable at Ϫ15 to Ϫ25°C through the expiration date printed on the label.
Reagent
Storage and stability
TUNEL reaction mixtureThe TUNEL reaction mixture should be pre-pared immediately before use and should not be stored.
Keep TUNEL reaction mixture on ice until use.Converter-POD
Once thawed the Converter-POD solution should be stored at 2–8°C (maximum stabil-ity 6 months).
Note: Do not freeze!
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2.1Product overview, continued
Please refer to the following table.
Benefit
SensitiveSpecificFastConvenient
Feature
Detection of apoptotic cell death at single cell level at very early stages (1, 2, 6).Preferential labeling of apoptosis versus necrosis (3, 4).
Short assay time (2-3 h).
•Reagents are provided in stable, opti-mized form.
•No dilution steps required.
•Suitable for fixed cells and tissue. This allows accumulation, storage and trans-port of samples (2, 5).
•Double staining enables identification of type and differentiation state of cells undergoing apoptosis (6).Every lot is function-tested on apoptotic cells in comparison to a master lot.
Advantage
Flexible
Function-tested
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2.2 Background informationCell death
Apoptosis
Two distinct modes of cell death, apoptosis and necrosis, can be distin-guished based on differences in morphological, biochemical and
molecular changes of dying cells.
Programmed cell death or apoptosis is the most common form of eukaryotic cell death. It is a physiological suicide mechanism that pre-serves homeostasis, in which cell death naturally occurs during normal tissue turnover (8, 9). In general, cells undergoing apoptosis display a characteristic pattern of structural changes in nucleus and cytoplasm, including rapid blebbing of plasma membrane and nuclear disintegra-tion. The nuclear collapse is associated with extensive damage to chromatin and DNA-cleavage into oligonucleosomal length DNA frag-ments after activation of a calcium-dependent endogenous endonu-clease (10, 11). However, very rare exceptions have been described where morphological features of apoptosis are not accompanied with oligonucleosomal DNA cleavage (37).
Apoptosis is essential in many physiological processes, including maturation and effector mechanisms of the immune system (12, 13), embryonic development of tissue, organs and limbs (14), development of the nervous system (15, 16) and hormone-dependent tissue remodeling (17). Inappropriate regulation of apoptosis may play an important role in many pathological conditions like ischemia, stroke, heart disease, cancer, AIDS, autoimmunity, hepatotoxicity and degen-erative diseases of the central nervous system (18–20).
In oncology, extensive interest in apoptosis comes from the observa-tion, that this mode of cell death is triggered by a variety of antitumor drugs, radiation and hyperthermia, and that the intrinsic propensity of tumor cells to respond by apoptosis is modulated by expression of several oncogenes and may be a prognostic marker for cancer treat-ment (21).
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continued2.2 Background information,
Identification of
apoptosis
Several methods have been described to identify apoptotic cells (22– 24). Endonucleolysis is considered as the key biochemical event of apoptosis, resulting in cleavage of nuclear DNA into oligonucleosome-sized fragments. Therefore, this process is commonly used for detec-tion of apoptosis by the typical “DNA ladder“ on agarose gels during electrophoresis. This method, however, can not provide information regarding apoptosis in individual cells nor relate cellular apoptosis to histological localization or cell differentiation.
This can be done by enzymatic in situ labeling of apoptosis induced DNA strand breaks. DNA polymerase as well as terminal deoxynucle-otidyl transferase (TdT) (1-6, 25-36, 41) have been used for the incor-poration of labeled nucleotides to DNA strand breaks in situ. The tailing reaction using TdT, which was also described as ISEL (in situ end labeling) (5, 35) or TUNEL (TdT-mediated dUTP nick end labeling) (1, 6, 31, 33) technique, has several advantages in comparison to the in situ nick translation (ISNT) using DNA polymerase:
•Label intensity of apoptotic cells is higher with TUNEL compared to ISNT, resulting in an increased sensitivity (2, 4).
•Kinetics of nucleotide incorporation is very rapid with TUNEL com-pared to the ISNT (2, 4).
•TUNEL preferentially labels apoptosis in comparison to necrosis, thereby discriminating apoptosis from necrosis and from primary DNA strand breaks induced by antitumor drugs or radiation (3, 4).
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3. Procedures and required materials
The working procedure described below has been developed and published by R. Sgonc and colleagues (6). The main advantage of this simple and rapid procedure is the use of fluorescein-dUTP to label DNA strand breaks. This allows the detection of DNA fragmentation by fluorescence microscopy directly after the TUNEL reaction prior to the addition of the secondary anti-fluorescein-POD-conjugate.
3.1Flow chart
The assay procedure is explained in the following flow chart.Adherent cells, cell smears and
cytospinpreparations
↓
Fixation↓Permeabilisation of samples
↓
Addition of TUNEL reaction mixture
↓OPTIONAL: Analysis of samples by fluorescence microscopy
↓
Addition of Converter-POD
↓Addition of Substrate solution
↓
Analysis of samples by light microscopy
Cryopreserved tissue sections
Paraffin-embeddedtissue sections↓
•Dewaxation •Rehydration•Protease treatment
↓Assay procedure
↓
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3.23.2.1
Preparation of sample material
Adherent cells, cell smears and cytospin preparations
Additional solutions required
Procedure
•Washing buffer: Phosphate buffered saline (PBS)•Blocking solution: 3% HFixation solution: 4% Paraformaldehyde in PBS, pH 72O2 in methanol
•.4, freshly pre-pared
•Permeabilisation solution: 0.1% Triton1) X-100 in 0.1% sodium citrate, freshly prepared (6)In the following table describes the fixation of cells, blocking of endo-genous peroxidase and cell permeabilisation.
Note: Fix and permeabilisate two additional cell samples for the nega-tive and positive labeling controls.StepAction
1Fix air dried cell samples with a freshly prepared Fixation solution for 1 h at 15-25°C.2Rinse slides with PBS.
3Incubate with Blocking solution for 10 min at 15-25°C.4Rinse slides with PBS.
5Incubate in Permeabilisation solution for 2 min on ice (2-8°C).
6
Proceed as described under 3.3.
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3.2.2 Tissue sections
3.2.2.1 Treatment of paraffin-embedded tissuePretreatment ofparaffin embeddedtissue
Tissue sections can be pretreated in 4 different ways. If you use Pro-teinase K the concentration, incubation time and temperature have to be optimized for each type of tissue (1, 29, 33, 36, 40, 42).
Note: Use Proteinase K only from Roche Applied Science, because it is tested for absence of nucleases which might lead to false-positive results!
The other 3 alternative procedures are also described in the following table (step 2).
•Xylene and ethanol (absolute, 95%, 90%, 80%, 70%, diluted in dou-Additional
ble distilled water)solutions required
•Washing buffer: PBS
•Proteinase K, nuclease free (Cat. No. 745 723), working solution: [10-20 g/ml in 10 mM Tris/HCl, pH 7.4-8] Alternative treatments•Permeabilisation solution: 0.1% Triton1) X–100, 0.1% sodium citrate, freshly prepared
•Pepsin* (0.25% - 0.5% in HCl, pH 2) or trypsin*, 0.01 N HCl, nuclease free
•0.1 M Citrate buffer, pH 6 for the microwave irradiation
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3.2.2.1 Treatment of paraffin-embedded tissue, continuedProcedure
In the following table the pretreatment of paraffin-embedded tissue with Proteinase K treatment and 3 alternative procedures are described.
Note: Add additional tissue sections for the negative and positive labeling controls.
StepAction1Dewax and rehydrate tissue section according to standard protocols (e.g. by heating at 60°C followed by washing in xylene and rehydration through a graded series of ethanol and double dist. water) (1, 33, 36).2Incubate tissue section for 15-30 min at 21–37°C with Pro-teinase K working solution.
Alternatives:Treatment:1. Permeabilisa-Incubate slides for 8 min.tion solution
2. Pepsin* (30, 40) 15-60 min at 37°C.or trypsin* 3. Microwave •Place the slide(s) in a plastic jar irradiationcontaining 200 ml 0.1 M Citrate
buffer, pH6.0.
•Apply 350 W microwave irradiation for 5 min.34
Rinse slide(s) twice with PBS.Proceed as described under 3.3.
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3.2.2.2Treatment of cryopreserved tissue
Additional •Fixation solution: 4% Paraformaldehyde in PBS, pH 7.4, freshly pre-solutions requiredpared
•Washing buffer: PBS
•Blocking solution: 3% H2O2 in methanol
•Permeabilisation solution (0.1% Triton1) X–100, 0.1% sodium citrate), freshly preparedCryopreserved tissue
In the following table the pretreatment of cryopreserved tissue is described.
Note: Fix and permeabilisate two additional samples for the negative and positive labeling controls.Step12
Action
Fixtissue section with Fixation solution for 20 min at 15–25°C.
Wash 30 min with PBS.
Note: For storage, dehydrate fixed tissue sections 2 min in absolute ethanol and store at Ϫ15 to Ϫ25°C.
Incubate with Blocking solution for 10 min at 15–25°C.Rinse slides with PBS.
Incubate in Permeabilisation solution for 2 min on ice (2–8°C).
Proceed as described under 3.3.
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3.33.3.1
Labeling protocolBefore you begin
One pair of tubes (vial 1: Enzyme Solution, and vial 2: Label Solution) is sufficient for staining 10 samples by using 50 l TUNEL reaction mix-ture per sample and 2 negative controls by using 50 l Label Solution per control.
Note: The TUNEL reaction mixture should be prepared immediately before use and should not be stored. Keep TUNEL reaction mixture on ice until use.Step12
Action
Remove 100 l Label Solution (vial 2) for two negative con-trols.
Add total volume (50 l) of Enzyme solution (vial 1) to the remaining 450 l Label Solution in vial 2 to obtain 500 l TUNEL reaction mixture.
Mix well to equilibrate components.
Preparation of TUNEL reaction mixture
3
•Micrococcal nuclease or Additional
reagents required•DNase I, grade I (Cat. No. 104 132)Controls
Two negative controls and a positive control should be included in each experimental set up.Negative control:Positive control:
Incubate fixed and permeabilized cells in 50 l/well Label Solution (without terminal transferase) instead of TUNEL reaction mixture.
Incubate fixed and permeabilized cells with micro-coccal nuclease or DNase I, grade I (3000 U/ml– 3 U/ml in 50 mM Tris-HCl, pH 7.5, 10 mM MgCl2 1mg/ml BSA) for 10 min at 15-25°C to induce DNA strand breaks, prior to labeling procedures.
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3.3.2
Labeling protocol for adherent cells, cell smears, cytospin preparations and tissues
•Washing buffer: PBSAdditional
equipment and •Humidified chambersolutions required
•Parafilm or coverslipProcedure
Please refer to the following table.Step123
Dry area around sample.
Add 50l TUNEL reaction mixture on sample.
Note: For the negative control add 50 l Label solution each. Toensure a homogeneous spread of TUNEL reaction mixture across cell monolayer and to avoid evaporative loss, samples should be covered with parafilm or coverslip during incuba-tion.
Add lid and incubate for 60 min at 37°C in a humidified atmo-sphere in the dark.
Rinse slide 3 times with PBS.
Samples can be analyzed in a drop of PBS under a fluores-cence microscope at this state. Use an excitation wavelength in the range of 450–500 nm and detection in the range of 515–565 nm (green).
Action
Rinse slides twice with PBS.
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3.3.3 Labeling protocol for difficult tissue
Additional •Citrate buffer, 0.1 M, pH 6.0.equipment and •Washing buffer: PBSsolutions required
Procedure
•Tris-HCl, 0.1 M pH 7.5, containing 3% BSA and 20% normal bovine serum•Plastic jar•Microwave
•Humidified chamberPlease refer to the following table.StepAction
1Dewax paraformaldehyde- or formalin-fixed tissue sections according to standard procedures.
2Place the slide(s) in a plastic jar containing 200 ml 0.1 M Citrate buffer, pH 6.0.
3
•Apply 750 W (high) microwave irradiation for 1 min.•Cool rapidly by immediately adding 80 ml double dist. water (20–25°C).
•Transfer the slide(s) into PBS (20–25°C).DO NOT perform a proteinase K treatment!
4
Immerse the slide(s) for 30 min at 15–25°C in Tris-HCl, 0.1 M pH 7.5, containing 3% BSA and 20% normal bovine serum.
5Rinse the slide(s) twice with PBS at 15–25°C.Let excess fluid drain off.
6Add 50 µl ofTUNEL reaction mixture on the section and.Note: For the negative control add 50 µl Label solution.7Incubate for 60 min at 37°C in a humidified atmosphere in the dark.
8
•Rinse slide(s) three times in PBS for 5 min each.
•Samples can be analyzed in a drop of PBS under a fluores-cence microscope at this state. Use an excitation wave-length in the range of 450–500 nm and detection in the range of 515–565 nm (green).
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3.4 Signal conversion Additional •equipment and •solutions required
•••Procedure
Washing buffer: PBSHumidified chamberParafilm or coverslip
DAB Substrate* (Cat. No. 1 718 096) or alternative POD substrateMounting medium for light microscopy
Please refer to the following table.Step12
Dry area around sample.
Add 50l Converter-POD (vial 3) on sample.
Note: To ensure a homogeneous spread of Converter-POD across cell monolayer and to avoid evaporative loss, samples should be covered with parafilm or cover slip during incuba-tion.
Incubate slide in a humidified chamber for 30 min at 37°C.Rinse slide 3× with PBS.
Add 50–100 l DAB Substrate or alternative POD substrates.Incubate slide for 10 min at 15–25°C.Rinse slide 3× with PBS.
Mount under glass coverslip (e.g. with PBS/glycerol) and ana-lyze under light microscope.
Alternative: Samples can be counterstained prior to analysis by light microscope.
Action
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4. Appendix
4.1 Trouble-shooting
This table describes various troubleshooting parameters.
Problem
Step/Reagent of Procedure
Possible cause
RecommendationTry different embedding material or different polymerization reagent.
NonspecificEmbedding of tissueUV-irradiation for labelingpolymerization of
embedding material (e.g. methacrylate) leads to DNA strand breaks
Fixation
Acidic fixatives (e.g. methacarn, Carnoy’s fixative)
•Try 4% buffered paraformal-dehyde.
•Try formalin or glutaralde-hyde.
TUNEL reaction
TdT concentration too Reduce concentration of TdT by highdiluting it 1:2 up to 1:10 with
TUNEL Dilution Buffer (Cat. No. 1 966 06).Endogenous POD activity
Block endogenous POD by immersing for 10 min in 3% H2O2 in methanol prior to cell permeabilisation.
•Block with normal anti-sheep serum.
•Block for 20 min with PBS containing 3% BSA.
•Reduce concentration of converter solution to 50%.
Converter solution
Non-specific binding of anti-fluorescein-POD
Nucleases
•Fix tissue immediately after Some tissues (e.g.
smooth muscles) organ preparation.show DNA strand •Perfuse fixative through liver breaks very soon after vein.tissue preparationSome enzymes are still active
Block with a solution containing ddUTP and dATP.
continued on next page
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4.1Trouble-shooting, continued
Step/Reagent of ProcedureFixation
Possible cause
Recommendation
ProblemHigh back-ground
Formalin fixation leads Try methanol for fixation but to a yellowish staining take into account that this might of cells containing lead to reduced sensitivity.melanin precursorsConcentration of labeling mix is too high for mamma car-cinoma
Endogenous POD activity
Reduce concentration of label-ing mix to 50% by diluting with TUNEL Dilution Buffer (Cat. No. 1 966 006).
Block endogenous POD by immersing for 10 min in 3% H2O2 in methanol prior to cell permeabilisation.
•Block with normal anti-sheep serum.
•Block for 20 min with PBS containing 3% BSA.
•Reduce concentration of converter solution to 50%.
TUNEL reaction
Converter solution
Non-specific binding of anti-fluorescein-POD
Sample
Mycoplasma contami-Mycoplasma detection Kit (Cat. nationNo. 1 296 7449).Highly proliferating cells
Double staining e.g. with
Annexin-V-Fluos (Cat. No. 1 828 681).
Note: Measuring via microplatereader not possible because of too high background.
Low labeling
Fixation
Ethanol and methanol •Try 4% buffered paraformal-can lead to low label-dehyde.
ing (nucleosomes are •Try formalin or glutaralde-not cross-linked with hyde.proteins during fixa-tion and are lost dur-ing the procedure steps)
Extensive fixation leads to excessive crosslinking of pro-teins
•Reduce fixation time.
•Try 2% buffered paraformal-dehyde.
Permeabilisation
Permeabilisation too •Increase incubation time.short so that reagents •Incubate at higher tempera-ture (e.g. 15–25°C).can’t reach their tar-•Try Proteinase K (concentra-get molecules
tion and time has to be opti-mized for each type of tissue).
•Try 0.1 M sodium citrate at 70°C for 30 min.
continued on next page
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4.1Trouble-shooting, continued
Problem
Step/Reagent of Procedure
Possible causeRecommendation•Treat tissue sections after dewaxing with Proteinase K (concentration, time and temperature have to be opti-mized for each type of tis-sue).
•Try microwave irradiation at 370 W (low) for 5 min in 200ml 0.1 M Citrate buffer pH 6.0 (has to be optimized for each type of tissue).•For cryosections apply 3 U/ml DNaseI, grade I.
•For paraffin-embedded tissue sections apply 1500 U/ml DNase I, grade I.
•In general, use 1 U/ml
DNaseI, grade I, dissolved in 10 mM Tris-HCl pH 7.4 con-taining 10 mM NaCl, 5 mM MnCl2, 0.1 mM CaCl2, 25 mM KCl and incubate 30 min at 37°C.
•Alternative buffer 50 mM Tris- HCl pH 7.5 containing 1mM MgCl2 and 1 mg/ml BSA.•Counterstaining with 5% methyl green in 0,1 M veronal acetate, pH 4.0 or Hematoxi-lin is possible (43).
•Double-staining with propid-ium iodide is possible but only for detection of morpho-logical cell changes.
Paraffin-embeddingAccessibility for
reagents is too low
No signal on positive control
DNase treatment
Concentration of DNase is too low
Weak sig-nals
CounterstainingNot suitable dye
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Roche Applied Science
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Roche Applied Science
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Roche Applied Science
For further information please access our web-site address at:http://biochem.roche.com
or the Apoptosis special interest site:
http://www.roche-applied-science.com/apoptosis
*available from Roche Applied Science1)
Triton is a registered trademark of Rohm & Haas, Philadelphia, USA.Sold through an arrangement with ENZO DIAGNOSTICS, INC.Purchase of this product does not include any right of license to exploit this product commercially.
This product or the use may be covered by one or more ENZO patents, including the following:
U.S. Patent Nos. 4,711,955; 5,328,824; 5,449,767; 5,241,060; 4,994,373; and 5,175,269; EP 0 063 897 BI; EP 0 117 440 BI;
EP 0 122 614 BI; and EP 0 128 332 BI; and Canadian Patent Nos. 1,219,824; 1,223,831; 1,309,672; 1,254,525; and 1,228,811.
25
Roche Applied Science
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