Apoptosis and Cell Proliferation (522915), страница 27
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Wherever possible, theanswers will direct you to pages and sections of this guide which can provide moreinformation.1Can I determine the number of apoptotic cells using the Cell Death Detection ELISAPLUS?A: No. The ELISA data is interpreted as achange in the level of death in an apoptotic population compared to an uninduced control population. It does notprovide data on individual cells.2What is the best way to get rid of nonspecific (false-positive) background inthe TUNEL (In Situ Cell Death Detection) kits?A: The best approach to reducing background depends on the results you obtain with the controls:P If cells incubated with fluoresceindUTP but without terminal transferase are false positive, try washingthe cells more thoroughly, reducingthe concentration of fluoresceindUTP, or using an alternative permeabilization procedure.AppendixP If false positives are produced onlyin reactions which include both fluorescein-dUTP and terminal transferase, the best means of reducingfalse positives is a reduction in enzyme concentration or a change inpermeabilization procedure.3What types of sample can be assayedwith the TUNEL method?A: Tissue sections, adherent cell cultures,cytospins and cell smears have all beenused with this assay (page 24, Section1.2.2.1).
Note, however, that the samplematerial must be preserved with a crosslinking fixative (such as paraformaldehyde).4Why isn’t substrate included in theTUNEL kits (In Situ Cell Death Detection Kits, AP or POD)?A: These kits will work with a variety ofcommon alkaline phosphatase or peroxidase substrates. Since many laboratories already have these substrates, andknow how these substrates work in“their” system we decided to leave themout. In addition this gives the researcherthe flexibility for secondary staining.5How long and at what temperature canI store my samples before analyzingthem with the various kits that youoffer?A: Table 24 gives some general guidelinesfor sample storage. Note however thatsome samples may be more or less stablethan others.6Is a special wash/stop buffer requiredfor the TUNEL kits?A: Our procedure does not require anequilibration buffer. Our wash buffer isPBS, a commonly used solution.KitStorage of samples before assayCell Death Detection ELISAPLUSPurified cytoplasmic samples can be stored at –20°C for2 weeks (with some reduction of signal)Apoptotic DNA Ladder KitPurified DNA can be stored at –20°C for 1 yearAnnexin-V-FLUOS Staining Kit; Annexin V-BiotinCells must be used live, directly after induction ofapoptosisCellular DNA Fragmentation ELISAPurified cytoplasmic samples can be stored at –20°C forat least 2 weeksG Table 24: Storage of samples for apoptosis assay1043Technical tipsTechnical tips on the TUNEL method3.1.2 Technical tips on the TUNELmethod3.1.2.2 TUNEL protocol for tissues whichtend to give false positives3.1.2.1 TUNEL: Improvement andevaluation of the method for in situapoptotic cell identification[from Dr.
Georg Fertig, Roche Molecular Biochemicals]Note: This is a summary of an article thatappeared in the Boehringer Mannheim Biochemica newsletter [Biochemica No. 2(1997)]. For further experimental detail andbackground, see the full Biochemica article.Summary: TUNEL or terminal deoxynucleotidyl transferase-mediated dUTPnick end-labeling, is a preferred method forrapid identification and quantification ofthe apoptotic cell fraction in cultured cellpreparations.
However, the accessibility ofDNA breaks to enzymatic reactions is reduced by the nuclear protein environment(Kerrigan et al., 1987) and impaired by cellfixation (Gold et al., 1994) and postfixation(Gorczyca et al., 1994). Thus, several sample pretreatments have been devised to improve TUNEL sensitivity (Desjardins andMacManus, 1995; Kerrigan et al., 1987;Lucassen et al., 1995).
An optimizedTUNEL protocol for cultured cells hasbeen developed.Sample:Paraffin-embedded tissue sections (e.g., of rabbit endometrium)Reagents: In Situ Cell Death DetectionKit, POD, Cat. No. 1 684 817DAB Substrate,Cat. No. 1 718 0961Dewax paraformaldehyde- or formalin-fixed tissue sections according tostandard procedures.2Place the slide(s) in a plastic jar containing 200 ml 0.1 M citrate buffer, pH6.0, put the jar in a microwave oven,and apply 750 W (high) microwave irradiation for 1 min. For rapid cooling,immediately add 80 ml redist. water(20°–25°C) to the jar, then transfer theslide(s) into PBS (20°–25°C).Caution: DO NOT perform a proteinase K treatment!3Immerse the slide(s) for 30 min at roomtemperature (RT) in a blocking solution containing 0.1 M Tris-HCl, 3%BSA, and 20% normal bovine serum,pH 7.5.Appendix[from Adrien Negoescu, Philippe Lorimier, FrancoiseLabat-Moleur, Laurent Azoti, Catherine Robert,Christiane Guillermat, Christian Brambilla, and ElisabethBrambilla; of Groupe de recherche sur le cancer dupoumon, Institut Albert Bonniot, Faculte de Medecine,Domaine de la Merci, 38706 Grenoble cedex, France,and Laboratoire de Pathologie cellulaire, CHRU, BP 217X,38043 Grenoble cedex 09, France]The protocol given below has been foundto eliminate the TUNEL labeling “false positives” seen with certain paraffin-embedded tissue sections (for example, of rabbitendometrium).
The key step is pretreatment of the slide with microwave irradiation rather than proteinase K.1053Technical tipsTUNEL protocol for tissues which tend to give false positives4Rinse the slide(s) twice with PBS at RT.Let excess fluid drain off.5Apply 50 µl of TUNEL reaction mixture to the section and incubate for60 min at 37°C in a humidified atmosphere.6Appendix73106Rinse slide(s) three times in PBS (5 minfor each wash).Note: At this stage, you can evaluate thesection under a fluorescence microscope.Block endogenous peroxidase activityby incubating slides for 10 min at RTwith 0.3% H2O2 in methanol.8Repeat steps 3 and 4 to block nonspecific binding of the anti-fluorescein-antibody to the tissue.9Add 50 µl Converter-POD, pre-diluted 1:2 in blocking solution (fromStep 3), and incubate for 30 min at 37°Cin a humidified atmosphere.10Rinse slide(s) three times in PBS at RTfor 5 min each.11Add 50 µl DAB substrate solution andincubate for 1–3 min at RT.12Wash slide(s) extensively in tap waterand counterstain if needed.Technical tipsTips for avoiding or eliminating potential TUNEL labeling artifacts3.1.2.3 Tips for avoiding or eliminating potential TUNEL labeling artifactsTo avoid this artifactWhich may be caused byNonspecific TUNEL labelingP DNA strand breaks induced by UV irradiation during tissue P Use a different embedding material, which does notembedding (UV used to polymerize tissue embeddingmaterial such as methacrylate)P Acid tissue fixatives (e.g., mathacarn, Carnoy’s fixative)Try the followingrequire UV irradiationP Use an alternate polymerization methodP Use buffered 4% paraformaldehyde as fixativecause DNA strand breaksP Endogenous nuclease activity which occurs soon aftertissue preparation (e.g., in smooth muscle tissue slices)P TdT concentration too high during TUNEL labelingP Fix tissue immediately after organ harvestP Perfuse fixative through liver vein in intact animalP Reduce concentration of TdT by diluting it 1:2 or 1:3 withTUNEL Dilution Buffer (Cat.No.
1966 006) containing30 mM Tris (pH 7.2) containing 140 mM sodium cacodylate and 1 mM CoCl2P Endogenous alkaline phosphatase activity duringconverter reactionP Block endogenous AP activity by adding 1 mM levamisoleto the AP substrate solutionP Endogenous peroxidase activity during converter reaction P Before permeabilizing cells, block endogenous PODactivity by immersing the slides in a solution of 0.3% H202in methanolP Nonspecific binding of anti-fluorescein antibodyconjugate during converter reactionP Block nonspecific sites with normal anti-sheep serumP Block nonspecific sites with PBS containing 3% BSA(20 min)P Use 1:2 dilution of converter solution in PBSP Formalin fixation, which causes yellow staining of cellscontaining melanin precursorsP TUNEL labeling mix too concentrated (e.g., forcarcinomas)P Endogenous alkaline phosphatase activity duringconverter reactionP Use methanol fixationNote: This fixation may lead to a reduction in TUNELlabeling sensitivityP Reduce concentration of labeling mix by diluting it 1:2with TUNEL Dilution Buffer (Cat.
No. 1966 006) containing30 mM Tris (pH 7.2) containing 140 mM sodium cacodylate and 1 mM CoCl2P Block endogenous AP activity by adding 1 mM levamisoleto the AP substrate solutionP Endogenous peroxidase activity during converter reaction P Before permeabilizing cells, block endogenous PODactivity by immersing the slides in a solution of 0.3% H202in methanolP Nonspecific binding of anti-fluorescein antibodyconjugate during converter reactionP Block nonspecific sites with normal anti-sheep serumP Block nonspecific sites with PBS containing 3% BSA(20 min)P Use 1:2 dilution of converter solution in PBSAppendixHigh background1073Technical tipsTips for avoiding or eliminating potential TUNEL labeling artifactsTo avoid this artifactWhich may be caused byTry the followingLow TUNEL labeling(low sensitivity)P Ethanol and methanol fixationP Use buffered 4% paraformaldehyde as fixativeP Extensive crosslinking during prolonged fixation reactions P Reduce fixation timeP Use buffered 2% paraformaldehyde as fixativeP Insufficient permeabilization of cells, so TUNEL reagentscannot reach nucleiP Pretreat with proteinase K (concentration and time mustbe optimized empirically)Note: To avoid possible nuclease contamination, use onlyProteinase K from Roche Molecular Biochemicals,Cat.
No. 161 519P Pretreat with 0.01 M sodium citrate for 30 min at 70°CP Increase TUNEL incubation timeP Restricted access of TUNEL reagents to nuclei, caused by P After dewaxing tissue sections, treat with proteinase Kparaffin-embeddingNo signal on positive controlP Inadequate DNase treatment (DNase concentrationtoo low)(concentration, time, and temperature must be optimizedempirically)Note: To avoid possible nuclease contamination, use onlyProteinase K from Roche Molecular Biochemicals,Cat. No 161 519P Immerse dewaxed tissue sections in 200 ml 0.01 Mcitrate buffer (pH 6.0) and treat with microwaveirradiation (370 W, 5 min)Note: Conditions must be experimentally optimized foreach tissueP For cryosections, apply 1 µg/ml DNaseP For paraffin-embedded tissue sections, apply 0.5 mg/mlDNaseP For many other samples, apply 1 U/ml DNase in a solutionof 10 mM Tris-HCl (pH 7.4), 10 mM NaCl, 5 mM MnCl2,0.1 mM CaCl2, 25 mM KCl; incubate 30 min at 37°CP As an alternative DNase buffer, use a solution of 10 mMTris-HCl (pH 7.5), 1 mM MgCl2, 1 mg/ml BSADiminished TUNEL stainingduring DNA counterstainingP Quenching of fluorescein signal by propidium iodide (PI)P Use 0.5 µg/ml PI as DNA stainP Substitute TO-PRO-3 (from Molecular Probes) in placeAppendixof PI3108Technical tipsTechnical tips3.1.3 Technical tips on the use ofAnnexin-V-Biotin for light microscopedetection3.1.4 Technical tips on the use of theApoptotic DNA Ladder Kit on tissuesamplesThe following protocol provides a methodfor the detection of Annexin-V-Biotinbinding to cell culture cells with lightmicroscopy.
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