213 SECTION II: ANTIBODYAPPLICATIONS 212 ForResearchUseOnly. NotForUse inDiagnosticProcedures. Seepg XXX forApplication,Reactivity andPathwayDiagram keys. CHAPTER12: IMMUNOHISTOCHEMISTRY (IHC) IHCPara nProtocol (usingSignalStain ® BoostDetectionReagent) *IMPORTANT: Please refer to theApplications section on the front page of product datasheet or productwebpage to determine whether a product is validated and approved for use on para n-embedded (IHC-P) tissue sections. Please see product datasheet or productwebpage for appropriate antibody dilution and unmasking solution. A.SolutionsandReagents NOTE: Prepare solutionswith reverse osmosis deionized or equivalent gradewater (dH 2 O). 1. Xylene 2. Ethanol, anhydrous denatured, histological grade (100% and 95%) 3. Deionizedwater (dH 2 O) 4. Hematoxylin (optional) 5.WashBuer: 1X TrisBuered Salinewith Tween ® 20 (TBST): To prepare 1 L 1X TBST: add100ml 10X TBST (#9997) to 900ml dH 2 O,mix. 6.AntibodyDiluentOptions: * a.SignalStain ® AntibodyDiluent: (#8112) b.TBST/5%normalgoat serum: To5ml 1X TBST, add250 µlNormalGoatSerum (#5425). c.PBST/5%normalgoat serum: To 5ml1XPBST, add250 µlNormalGoatSerum (#5425). 1XPhosphateBueredSaline (PBS): To prepare 1 L1X PBS: add50ml 20X PBS (#9808) to 950ml dH 2 O,mix. 1XPBS/0.1%Tween ® 20 (PBST): To prepare 1 L 1X PBST: add 1ml Tween ® 20 to 1 L1XPBS andmix. 7.AntigenUnmaskingOptions: * a.Citrate: 10mM SodiumCitrateBuer: To prepare 1 L, add2.94 g sodium citrate trisodium salt dihydrate (C 6 H 5 Na 3 O 7 •2H 2 O) to 1 L dH 2 O.Adjust pH to 6.0. b.EDTA: 1mM EDTA: To prepare 1 L add0.372 g EDTA (C 10 H 14 N 2 O 8 Na 2 •2H 2 O) to 1 L dH 2 O.Adjust pH to 8.0. c.TE: 10mM Tris/1mM EDTA, pH 9.0: To prepare1 L, add 1.21 g Tris base (C 4 H 11 NO 3 ) and 0.372 g EDTA (C 10 H 14 N 2 O 8 Na 2 •2H 2 O) to950ml dH 2 O.Adjust pH to 9.0, then adjust final volume to 1 Lwith dH 2 O. d.Pepsin: 1mg/ml in Tris-HCl, pH 2.0. 8.3%HydrogenPeroxide: To prepare 100ml, add10ml 30%H 2 O 2 to 90ml dH 2 O. 9.BlockingSolution: TBST/5%NormalGoatSerum: to5ml 1X TBST, add 250 µlNormalGoat Serum (#5425). 10.DetectionSystem: SignalStain ® Boost IHCDetectionReagents (HRP,Mouse #8125;HRP,Rabbit #8114 ) 11.Substrate: SignalStain ® DABSubstrate Kit (#8059). B.Deparanization/Rehydration NOTE: Do not allow slides to dry at any time during this procedure. 1.Deparanize/hydrate sections: a. Incubate sections in3washes of xylene for 5min each. b. Incubate sections in 2washes of 100% ethanol for 10min each. c. Incubate sections in 2washes of 95% ethanol for 10min each. 2. Wash sections2 times in dH 2 O for 5min each. C.AntigenUnmasking* NOTE: Consult product datasheet for specific recommendation for the unmasking solution/protocol. 1.ForCitrate: Bring slides to a boil in 10mM sodium citrate buer, pH6.0;maintain at a sub-boiling temperature for 10min. Cool slides on bench top for 30min. 2.ForEDTA: Bring slides to a boil in 1mM EDTA, pH8.0: followwith 15min at a sub-boiling temperature.No cooling is necessary. 3.ForTE: Bring slides to a boil in10mM Tris/1mM EDTA, pH9.0: thenmaintain at a sub-boiling temperature for 18min. Cool at room temperature for30min. 4.ForPepsin: Digest for 10min at 37°C. D.Staining NOTE: Consult product datasheet for recommended antibody diluent. 1. Wash sections in dH 2 O 3 times for 5min each. 2. Incubate sections in 3% hydrogen peroxide for10min. 3. Wash sections in dH 2 O 2 times for 5min each. 4. Wash sections inwash buer for5min. 5. Block each sectionwith100–400 µl blocking solution for1 hr at room temperature. 6. Remove blocking solution and add 100–400 µl primary antibody diluted in recommended antibody diluent to each section*. Incubate overnight at4°C. 7. Equilibrate SignalStain ® BoostDetectionReagent to room temperature. 8. Remove antibody solution andwash sectionswithwash buer three times for5min each. 9. Cover sectionwith1–3 dropsSignalStain ® BoostDetectionReagent as needed. Incubate in a humidified chamber for 30min at room temperature. 10. Wash sections three timeswithwash buer for 5min each. 11. Add 1 drop (30 µl)SignalStain ® DABChromogenConcentrate to 1ml SignalStain ® DABDiluent andmixwell before use. 12. Apply100–400 µl SignalStain ® DAB to each section andmonitor closely. 1–10min generally provides an acceptable staining intensity. 13. Immerse slides in dH 2 O. 14. If desired, counterstain sectionswith hematoxylin permanufacturer’s instructions. 15. Wash sections in dH 2 O two times for 5min each. 16.Dehydrate sections: a. Incubate sections in 95% ethanol two times for 10 sec each. b. Repeat in 100% ethanol, incubating sections two times for10 sec each. c. Repeat in xylene, incubating sections two times for10 sec each. 17. Mount sectionswith coverslips. Foroptimal results,alwaysuse the recommendedprimary antibodydiluent,as indicatedon theproductdatasheet. SignalStain ® AntibodyDiluent #8112: IHC analysis of para n- embedded human breast carcinoma (top) andHCC827 xenograft (bottom) using Phospho-Akt (Ser473) (D9E) XP ® RabbitmAb#4060 or Phospho- EGFReceptor (Tyr1173) (53A5)Rabbit mAb #4407 after dilution in either #8112 (left) or TBST/5%NGS (right). #4060 SignalStain ® #8112 TBST-5%NGS #4407 A superior signal is achieved when#4060 is diluted in #8112 as comparedwithTBST/5%NGS. No one diluent enables the optimal performance of all antibodies. IHCFrozenProtocol (usingSignalStain ® BoostDetectionReagent) IMPORTANT: Please refer to theApplications section on the front page of product datasheet or productwebpage to determine whether a product is validated and approved for use on frozen (IHC-F) tissue sections.Please see product datasheet or product webpage for appropriate antibody dilution. NOTE: Please see product datasheet andwebsite for product-specific protocol recommendations. A.SolutionsandReagents NOTE: Prepare solutionswith reverse osmosis deionized or equivalent gradewater (dH 2 O). 1. Xylene 2. Ethanol (anhydrous denatured, histological grade 100% and 95%) 3. Hematoxylin (optional) 4.1XPhosphateBueredSaline (PBS): To prepare1 L1X PBS: add 50ml 20X PBS (#9808) to 950ml dH 2 O,mix. 5.FixativeOptions: For optimal fixative, please refer to the product datasheet. a. 10% neutral buered formalin b. Acetone c. Methanol d.3% Formaldehyde: To prepare 100ml, add18.75ml 16% formaldehyde to 81.25ml1XPBS. 6.WashBuer: 1X TrisBuered Salinewith Tween ® 20 (TBS): To prepare 1 L 1X TBS: add 100ml 10X TBS (#12498) to900 ml dH 2 O,mix. 7.Methanol/Peroxidase: To prepare, add10ml 30%H 2 O 2 to 90mlmethanol. Store at -20°C. 8.BlockingSolution: 1X TBS/0.3% Triton ™ X-100/5%NormalGoatSerum (#5425). To prepare, add 500 µl goat serum and 30 µl Triton ™ X-100 to 9.5ml1X TBS. 9.DetectionSystem: SignalStain ® Boost IHCDetectionReagents (HRP,Mouse #8125;HRP,Rabbit #8114 ) www.cellsignal.com /cstprotocols Formore in-depth helpwith IHC, please see our IHCProtocols and Troubleshooting videos at www.cellsignal.com/ihcvideos IHCTips& Techniques Videos 255 SECTION III: WORKFLOWTOOLS 254 ForResearchUseOnly. NotForUse inDiagnosticProcedures. Seepg XXX forApplication,Reactivity andPathwayDiagram keys. How can IusePTM-specificproteomics toenablediscovery ofnoveldiseasedriversand identificationofbiomarkers? How can Iprofile changes inspecificPTMsitesonkey target proteins toexplore changes in criticalsignalingevents? Post-translationalmodifications such as phosphorylation, acetylation,methylation, ubiquitination, and others are critical regulators of protein activity and function.Understanding the role of PTMs in disease states and developing novel biomarkers and therapeutics are areas of intense research.However, researcherswho study PTMs are challenged by their inherent low levels,whichmake them dicult to identify and quantify fromwhole cell or native samples. This challenge can be overcome by using immunoanity enrichment to isolate specific peptides containingPTMs of interest from a protease-digested cell or tissue extract. LC-MS/MS analysis is then employed to identify and quantify the isolated peptides. PTMScan ® Technology, proprietary to CST, utilizes the specificity ofPTM-specific andmotif antibodies to enrich target peptides from the background of non-modified endogenous peptides, enabling the identification ofmodified proteins that otherwisemay not be detected.Hundreds to thousands of post-translationallymodified peptides can be identified and quantified in a single experiment. This approach can be used to studymodifications such as phosphorylation, acetylation,methylation, ubiquitination, succinylation, or cleaved caspase. The highly conserved nature ofPTMs and substratemotifs acrossmany dierent species alsomakes this approach applicable tomany dierentmodel systems. PTMSiteDiscoveryandProfiling • Discover candidate biomarkers linked to activation or repression of a specific class of PTMs. • Perform a comprehensive proteomic survey of thousands of PTM sites associatedwith awide variety of organisms or a diseasemodel system, integrating the results into known signaling pathways or helping to define novel signaling networks. • Detect substrates of novel signaling proteins (kinases, phosphatases, ubiquitin ligases, deubiquitinases, acetyl transferases,methyl transferases, or succinyl transferases). • Profile and quantify global eects of a candidate therapeutic on a specific type ofPTM, identifying nodes of interest for further study. • Understand how cross-talk among various PTMs (phosphorylation, ubiquitination, acetylation, methylation) is related to a particular biological response or involved in cell development or dierentiation. • Analyze downstream eects of targeted gene silencing on signaling and potential activation of alternative pathways. • Identify protein-protein interaction binding partners alongwith their corresponding PTMs. DiscoverLowAbundancePTMSites Immunoanity enrichment allows you to identify low abundancemodifications by selectively capturing the PTM in the context of themotif of interest from a population of endogenous peptides. For example, the specificity of immunoanity enrichment complements that of IMAC, a charge-basedmetal anity method commonly used for phospho-peptides. IMAC is driven by general coordination chemistry dictated by the immobilizedmetal ion (1) . IMAC can therefore lead to enrichment of peptides frommore abundant proteins for subsequent LC-MS/MS analysis, but not so readily for less abundant phospho-peptides. 19 CHAPTER19: DISCOVERY IdentificationofBiomarkers PTMScan Technology has been used to identify novel biomarkers in a number of dierentmodel sys- tems (2–6) .One example of the application of PTMScan Technologywas conducted in a large-scale survey of tyrosine kinase activity in lung cancer, performed atCST. For this study,we used a phospho- tyrosinemotif antibody to analyze changes in phosphorylation across the proteome in non-small cell lung carcinoma (NSCLC) cell lines and tissues. We surveyed the phospho-tyrosine status of receptor tyrosine kinases (RTK) and non-receptor tyrosine kinases in 41NSCLC cell lines and over 150NSCLC tumors.Over50 tyrosine kinases andmore than 2,500 downstream substrates that play roles inNSCLC growth and progressionwere identified. Two very exciting findings from this studywere the identification of novelALK andROS1C-terminal fusion proteins (EML4-ALK,CD74-ROS, SLC34A2-ROS) in someNSCLC cell lines and tumors (7) . Further investigation ofALK andROS1 in other cancers led to the identification of a novel FN1-ALK fusion protein in ovarian cancer and a FIG-ROS1 fusion in cholangiocarcinoma (8,9) . As one consequence of this study, a companion diagnostic immunohistochemistry (IHC) assay for NSCLCwas developed. In2011, the diagnostic IHC assaywas approved in theU.S. for patientswith tu- mor samples that stain positive forALK fusion protein expression for crizotinib treatment ofNSCLC (10) . A total of27,372 unique phospho- peptideswere isolated fromMKN-45 cells using both IMAC andmotif antibody enrichment strategies. In this study, distinct sets ofmodified peptideswere identified by the two anity enrichmentmethods,with only 5.6% overlap between theMS/ MS identified peptides. IMAC (13,892) 5.6% AllAntibody (13,480) Complementarymethods: immunoa nity and IMACenrichment forphospho-peptides. Discovery USINGPEPTIDE IMMUNOAFFINITYENRICHMENTANDLC-MS/MS TOSTUDYPOST-TRANSLATIONALMODIFICATIONS (PTMs) ALK (D5F3)XP ® RabbitmAb#3633: IHC analysis of paran-embedded human lung carcinomawith high (left) and low levels (right) ofALK fusion protein expression using #3633. ALK fusionprotein detection inhuman lung carcinoma Carcinoma-associated fusionproteins discoveredusingPTMScanTechnology Discoveryworkflow forphospho-tyrosine modifications altered inNSCLC Kinase FamilyTargeting LC-MS/MS and BioinformaticAnalysis Immunoprecipitation UsingMotifAntibody Cell orTissueSamples www.cellsignal.com /discovery 89 SECTION I: RESEARCHAREAS 88 ForResearchUseOnly. NotForUse inDiagnosticProcedures. Seepg XXX forApplication,Reactivity andPathwayDiagram keys. www.cellsignal.com /customurlgoeshere CHAPTER03: CELLGROWTHANDDEATH GAS2 ROCK1 Acinus DFF40 TNF-R1 TNF-R2 DR3 APO-3 DR4/5 Fas/ CD95 TRADD CYLD RIP TRADD TRADD RIP FasL APO-3L/ TWEAK APO-2L/ TRAIL RelB RelB p65/ RelA p52 IKK// IKK FADD Apaf1 XIAP XIAP MKK1 JNR RIP3 RIP1 RIP1 FADDRIP1 Casp-8, -10 Casp-8,-10 ASK1 LaminA Actin DFF45 PARP Casp-7 Casp-6 Casp-9 Casp-8 Casp-8 Casp-3 FADD FLIP TRAF2 TRAF3 cIAP1/2 cIAP1/2 cIAP1/2 FADD TRAF2 TRAF1 TRAF2 TRAF1 FADD Daxx IIa IIb TNF- TNF- Active Caspase-8 NIK IB IB Smac NF-B NF-B p100 NF-B p50 MLKL Bcl-2 Bcl-2 tBID BID ub CellShrinkage MembraneBlebbing DNA Fragmentation Chromatin Condensation Transcription Processing Apoptosis Survival Pro-Apoptotic Pro-Survival Apoptosis can be induced through the activation of death receptors including Fas, TNF α R,DR3,DR4, andDR5 by their respective ligands.Death receptor ligands characteris- tically initiate signaling via receptor oligomerization,which in turn results in the recruitment of specialized adaptor proteins and activation of caspase cascades.Binding of FasL induces Fas trimerization,which recruits initiator caspase-8 via the adaptor protein FADD.Caspase-8 then oligomerizes and is activated via autocatalysis.Activated caspase-8 stimulates apoptosis via two parallel cascades: it can directly cleave and activate caspase-3, or alternatively, it can cleaveBid, a pro-apoptoticBcl-2 family protein. Truncated Bid (tBid) translocates tomitochondria, inducing cytochrome c release,which sequentially activates caspase-9 and -3. TNF- α andDR-3L can deliver pro- or anti-apoptotic signals. TNF α R andDR3 promote apoptosis via the adaptor proteins TRADD/FADD and the activation of caspase-8. Interaction of TNF- α with TNF α Rmay activate theNF- κ B pathway viaNIK/IKK. The activation ofNF- κ B induces the expression of pro-survival genes includingBcl-2 and FLIP, the latter can directly inhibit the activation of caspase-8. FasL and TNF- α may also activate JNK viaASK1/MKK7.Activation of JNKmay lead to the inhibition ofBcl-2 by phosphorylation. In the absence of caspase activation, stimu- lation of death receptors can lead to the activation of an alternative programmed cell death pathway termed necroptosis by forming complex IIb. SelectReviews: Declercq,W., VandenBerghe, T., and Vandenabeele,P. (2009) Cell 138,229–232. • Fuchs, Y. andStellerH. (2011) Cell 147,742–758. • Kantari,C. andWalczak,H. (2011) Biochim.Biophys.Acta. 1813,558–563. • Kaufmann, T.,Strasser,A., and Jost, P.J. (2012) CellDeathDi er. 19,42–50. • Lavrik, I.N. and Krammer, P.H. (2012) CellDeathDi er. 19,36–41. • VanHerreweghe, F., Festjens,N.,Declercq,W., and Vandenabeele,P. (2010) Cell.Mol. Life Sci. 67,1567–1579. • Wajant,H. and Scheu- rich,P. (2011) FEBS J. 278,862–876. Casp-9 Casp-3 tBID tBID Bim Bmf Bmf BID Bax Bak Bax Bax Bad Bcl-2 Bcl-2 Bcl-2 Bcl-2 Puma Bcl-xL Bcl-xL PKC Erk1/2 p90RSK p70S6K Akt PKA 14-3-3 Bad 14-3-3 Bad Apaf-1 Cyto c Arts XIAP Smac/ Diablo AIF ATM/ ATR JNK JNK p53 PI3K Bim Casp-8,-10 FADD ub Bak Mcl-1 HECTH9 HECTH9 PIDD RAIDD CaMKII Hrk Hrk DLC2 LC8 HSP60 HtrA2 EndoG ING2 SirT2 ARD1 NatA Fas/ CD95 FasL Noxa Mcl-1 Calcineurin [NAD] Casp-2 Microtubules Acetyl-CoA Apoptosis DNADamage GenotoxicStress Cytosolic Sequestration Bcl-2Family SurvivalFactors: GrowthFactors,Cytokines, etc. DeathStimuli: SurvivalFactorWithdrawal Pro-Apoptotic Pro-Survival MitochondrialControlofApoptosis TheBcl-2 family of proteins regulate apoptosis by controllingmitochondrial permeability. The anti-apoptotic proteinsBcl-2 andBcl-xL reside in the outermitochondrialwall and inhibit cytochrome c release. The pro-apoptoticBcl-2 proteinsBad,Bid,Bax, andBimmay reside in the cytosol but translocate tomitochondria following death signaling, where they promote the release of cytochrome c.Bad translocates tomitochondria and forms a pro-apoptotic complexwithBcl-xL. This translocation is inhibited by survival factors that induce the phosphorylation ofBad, leading to its cytosolic sequestration.CytosolicBid is cleaved by caspase-8 following signaling through Fas; its active fragment (tBid) translocates tomitochondria.Bax andBim translocate tomitochondria in response to death stimuli, including survival factorwithdrawal.Activated followingDNA dam- age, p53 induces the transcription ofBax,Noxa, andPuma.Upon release frommitochondria, cytochrome c binds toApaf-1 and forms an activation complexwith caspase-9. Although themechanism(s) regulatingmitochondrial permeability and the release of cytochrome c during apoptosis are not fully understood,Bcl-xL,Bcl-2, andBaxmay influ- ence the voltage-dependent anion channel (VDAC),whichmay play a role in regulating cytochrome c release.Mule/ARF-BP1 is aDNA damage-activated E3 ubiquitin ligase for p53, andMcl-1, an anti-apoptoticmember ofBcl-2. SelectReviews: Brenner,D. andMak, T.W. (2009) Curr.Opin.CellBiol. 21,871–877. • Chalah,A.,Khosravi-Far,R. (2008) Adv. Exp.Med.Biol. 615,25–45. • Lindsay, J., Esposti,M.D., andGilmore,A.P. (2011) Biochim.Biophys.Acta. 1813,532–539. • Ola,M.S.,Nawaz,M., andAhsan,H. (2011) Mol.Cell.Biochem. 351, 41–58. • Pradelli, L.A.,Béné- teau,M., andRicci, J.E. (2010) Cell.Mol. LifeSci. 67,1589–1597. • Rong, Y. andDistelhorst,C.W. (2008) Annu.Rev. Physiol. 70, 73–91. • Speidel,D. (2010) Trends CellBiol. 20, 14–24. • Suen,D.F.,Norris, K.L., and Youle,R.J. (2008) GenesDev. 22, 1577–1590. DeathReceptorSignaling ©2003–2014Cell Signaling Technology, Inc. • Wewould like to thankProf. Junying Yuan,HarvardMedical School,Boston,MA, for reviewing this diagram. ©2002–2014CellSignaling Technology, Inc. • Wewould like to thankProf. Junying Yuan,HarvardMedical School,Boston,MA, for reviewing this diagram.