Skip to main content
SpringerLink
Log in

Optimization of Immunostaining for Prospective Image Analysis

  • Protocol
  • First Online:
Molecular Profiling

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1606))

Abstract

Biomarker discovery is a crucial part of the fast developing field of personalized medicine. Antibody-based techniques including immunostaining of tissue samples are widely used for biomarker evaluation in preclinical and clinical studies. When used in conjunction with robust image analysis methods, it provides a powerful means to assess biomarker modulation, toxicity, and patient response to targeted agents. Here, we describe the optimization of immunofluorescent (IF) staining protocols and a sample IF multiplex protocol suitable for colocalization image analysis.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 129.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 169.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Straus SE, Sackett DL (1999) Applying evidence to the individual patient. Ann Oncol 10:29–32

    Article  CAS  PubMed  Google Scholar 

  2. Khleif S, Doroshow J, Hait W (2010) N.AACR-FDA-NCI cancer biomarkers collaborative. AACR-FDA-NCI cancer biomarkers collaborative consensus report: advancing the use of biomarkers in cancer drug development. Clin Cancer Res 16:3299–3318

    Article  CAS  PubMed  Google Scholar 

  3. Walk E (2010) Improving the power of diagnostics in the era of targeted therapy and personalized healthcare. Curr Opin Drug Discov Devel 13:226–234

    CAS  PubMed  Google Scholar 

  4. Lee J, Devanarayan V, Barrett Y et al (2006) Fit-for-purpose method development and validation for successful biomarker measurement. Pharm Res 23:312–328

    Article  CAS  PubMed  Google Scholar 

  5. Kummar S, Kinders R, Gutierrez M et al (2009) Phase 0 clinical trial of poly (ADP-ribose) polymerase inhibitor ABT-888 in patients with advanced malignancies. J Clin Oncol 27:2705–2711

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Coons A, Creech H, Jones R (1941) Immunological properties of an antibody containing a fluorescent group. Proc Soc Exp Biol Med 47:200–202

    Article  CAS  Google Scholar 

  7. Dunstan R, Wharton K, Quigley C et al (2011) The use of immunocytochemistry for biomarker assessment-can it compete with other technologies? Toxicol Pathol 39:988–1002

    Article  PubMed  Google Scholar 

  8. Rizzardi A, Johnson A, Vogel R et al (2012) Quantitative comparison of immunohistochemical staining measured by digital image analysis versus pathologist visual scoring. Diagn Pathol 7:42–52

    Article  PubMed  PubMed Central  Google Scholar 

  9. Mosedale D, Metcalfe C, Grainger D (1996) Optimization of immunofluorescence methods by quantitative image analysis. J Histochem Cytochem 44:1043–1050

    Article  CAS  PubMed  Google Scholar 

  10. Wahlby C, Erlandsson F, Nyberg K et al (2001) Multiple tissue antigen analysis by sequential immunofluorescence staining and multi-dimensional image analysis. Proceeding of SCIA, pp 25–31

    Google Scholar 

  11. Molyneux G, Smalley M (2011) The cell of origin of BRCA1 mutation-associated breast cancer: a cautionary tale of gene expression profiling. J Mammary Gland Biol Neoplasia 16:51–59

    Article  PubMed  Google Scholar 

  12. Chen G, Gharib T, Huang C et al (2002) Discordant protein and mRNA expression in lung adenocarcinomas. Mol Cell Proteomics 4:304–313

    Article  Google Scholar 

  13. Cummings J, Ward T, Dive C (2010) Fit-for-purpose biomarker validation for anticancer drug development. Drug Discov Today 15:816–825

    Article  CAS  PubMed  Google Scholar 

  14. Mass R (2006) erb-B2 as a therapeutic target. In: Gasparini G, Hayes D (eds) Biomarkers of breast cancer. Humana Press, Towtowa, NJ, p 15973

    Google Scholar 

  15. Wolff A, Hammond M, Schwartz J et al (2007) American Society of Clinical Oncology/College of American Pathologists guideline recommendations for human epidermal growth factor receptor 2 testing in breast cancer. Arch Pathol Lab Med 131:18–43

    CAS  PubMed  Google Scholar 

  16. Petty H (2007) Fluorescence microscopy: established and emerging methods, experimental strategies, and applications in immunology. Microsc Res Tech 70:687–709

    Article  PubMed  Google Scholar 

  17. Spring K (2003) Fluorescence microscopy. In: Encyclopedia of optical engineering. Mercel and Dekker, New York, NY, pp 548–555

    Google Scholar 

  18. Michalet X, Kapanidis A, Laurence T et al (2003) The power and prospects of fluorescence microscopes and spectroscopies. Annu Rev Biophys Biomol Struct 32:161–182

    Article  CAS  PubMed  Google Scholar 

  19. Wahlby C, Erlandsson F, Bengtsson E et al (2002) Sequential immunofluorescence staining and image analysis for detection of large numbers of antigens in individual cell nuclei. Cytometry 47:32–41

    Article  CAS  PubMed  Google Scholar 

  20. Spruessel A, Steimann G, Jung M et al (2004) Tissue ischemia time affects gene and protein expression patterns within minutes following surgical tumor excision. BioTechniques 36:1030–1037

    CAS  PubMed  Google Scholar 

  21. Atkins D, Reiffen K, Tegtmeier C et al (2004) Immunohistochemical detection of EGFR in paraffin embedded tumor tissues: variation in staining intensity due to choice of fixative and storage time of tissue sections. J Histochem Cytochem 7:893–901

    Article  Google Scholar 

  22. Burns J, Li Y, Cheney C et al (2009) Choice of fixative is crucial to successful immunohistochemical detection of phosphoproteins in paraffin-embedded tumor tissues. J Histochem Cytochem 57:257–264

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Samaratunga H, Montironi R, True L et al (2011) International Society of Urological Pathology (ISUP) consensus conference on handling and staging of radical-prostatectomy specimens. Working group 1: specimen handling. Mod Pathol 24:5–15

    Article  Google Scholar 

  24. Best S, Sawyers Y, Fu V (2007) Integrity of prostatic tissue for molecular analysis after robotic-assisted laparoscopic and open prostatectomy. Urology 70:328–332

    Article  PubMed  PubMed Central  Google Scholar 

  25. Xie R, Chung J, Ylaya K et al (2011) Factors influencing the degradation of archival formalin-fixed paraffin-embedded tissue sections. J Histochem Cytochem 59:356–365

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Institute of Laboratory Animal Resources CoLS, National Research Council (1996) Guide for the care and use of laboratory animals. National Academy Press, Washington, D.C., p 125

    Google Scholar 

  27. Goldenthal K, Hedman K, Chen J et al (1985) Postfixation detergent treatment for immunofluorescence suppresses localization of some integral membrane proteins. J Histochem Cytochem 33:813–820

    Article  CAS  PubMed  Google Scholar 

  28. Golubeva Y, Rogers K (2009) Collection and preparation of rodent tissue samples for histopathological and molecular studies in carcinogenesis. Methods Mol Biol 511:3–60

    Article  CAS  PubMed  Google Scholar 

  29. Fox C, Johnson F, Whiting J et al (1985) Formaldehyde fixation. J Histochem Cytochem 33:845–853

    Article  CAS  PubMed  Google Scholar 

  30. Puchtler H, Meloan S (1985) On the chemistry of formaldehyde fixation and its effects on immunohistochemical reactions. Histochemistry 82:201–204

    Article  CAS  PubMed  Google Scholar 

  31. Hann B, Balmain A (2001) Building ‘validated’ mouse models of human cancer. Curr Opin Cell Biol 13:778–784

    Article  CAS  PubMed  Google Scholar 

  32. Atkinson B, Walden B (eds) (1985) Changes in eukaryotic gene expression in response to environmental stress. Imprint Orlando Academic, Orlando, p 379

    Google Scholar 

  33. Mori N, Mizuno D, Goto S (1978) Increase in ratio of 18S RNA to 28 S RNA in cytoplasm of mouse tissues during aging. Mech Ageing Dev 8:285–297

    Article  CAS  PubMed  Google Scholar 

  34. Ross J (1995) mRNA stability in mammalian cells. Microbiol Rev 59:423–450

    CAS  PubMed  PubMed Central  Google Scholar 

  35. Kabnick K, Housman D (1998) Determinants that contribute to cytoplasmic stability of human c-fos and beta-globin mRNAs are located at several sites in each RNA. Mol Cell Biol 8:8–13

    Google Scholar 

  36. Melan M, Sluder G (1992) Redistribution and differential extraction of soluble proteins in permeabilized cultured cells: implications for immunofluorescence microscopy. J Cell Sci 101:731–743

    PubMed  Google Scholar 

  37. Babic A, Loftin I, Stanislaw S et al (2010) The impact of pre-analytical processing on staining quality for H&E, dual hapten, dual color in situ hybridization and fluorescent in situ hybridization assays. Methods 52:287–300

    Article  CAS  PubMed  Google Scholar 

  38. Little E (2010) What difference does a difference make? Visions Conference. http://digitalpathologyassociation.org/_data/files/2010_Presentations/Little_-_What_Difference_Does_A_Difference_Make.ppt

  39. Taylor C, Levenson R (2006) Quantification of immunohistochemistry-issues concerning methods, utility and semiquantitative assessment II. Histopathology 49:411–424

    Article  CAS  PubMed  Google Scholar 

  40. Davis A, Richter A, Becker S et al (2014) Characterizing and diminishing autofluorescence in formalin-fixed paraffin-embedded human respiratory tissue. J Histochem Cytochem 62:405–423

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Myers J (2006) Antigen retrieval: a review of commonly used methods and devices. MLO Med Lab Obs 38:10–15

    Google Scholar 

  42. Shaner N, Steinbach P, Tsien R (2005) A guide to choosing fluorescent proteins. Nat Methods 2:905–909

    Article  CAS  PubMed  Google Scholar 

  43. Pawley J (1995) Handbook of biological confocal microscopy. Plenum Press, New York

    Book  Google Scholar 

  44. Fluorescence quantum yields (QY) and lifetimes (Τ) for Alexa fluor dyes–Table 1.5 | Thermo Fisher Scientific (2016) http://www.thermofisher.com. Accessed 31 Mar 2016

  45. Huang B (2012) Flourescent microscopy II. Fluorescent dyes. http://huanglab.ucsf.edu/Lectures/2012%20UCSF%20Fluorescence%20dyes.pdf. Accessed 31 Mar 2016

  46. Burry R (2010) Immunocytochemistry. Springer, New York

    Book  Google Scholar 

  47. Lakowicz J (2006) Principles of fluorescence spectroscopy, 3rd edn. Plenum Press, New York

    Book  Google Scholar 

  48. Khoury T, Sait S, Hwang H et al (2009) Delay to formalin fixation effect on breast biomarkers. Mod Pathol 22:1457–1467

    Article  CAS  PubMed  Google Scholar 

  49. Stack E, Wang C, Roman K et al (2014) Multiplexed immunohistochemistry, imaging, and quantitation: a review, with an assessment of tyramide signal amplification, multispectral imaging and multiplex analysis. Methods 70:46–58

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

This Research was supported (in part) by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research. This project has been funded in whole or in part with federal funds from the National Cancer Institute, National Institutes of Health, under Contract No. HHSN261200800001E. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the US government. The authors would like to thank staff of Clinical Pharmacodynamic Biomarkers Program, Applied/Developmental Research Directorate, and also Donna Butcher, Brad Gouker, and Simona Florea of Pathology-Histotechnology Laboratory, Leidos Biomedical Research, Inc., and Frederick National Laboratory for Cancer Research, USA, for technical assistance.

Author information

Authors and Affiliations

  1. Pharmacodynamics Assay Section (PADIS), Laboratory of Human Toxicology and Pharmacology, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Frederick, MD, USA

    Scott M. Lawrence M.S., H.T. (A.S.C.P.)

  2. MedImmune, Gaithersburg, MD, USA

    Yelena G. Golubeva Ph.D., H.T. (A.S.C.P.)

Authors
  1. Scott M. Lawrence M.S., H.T. (A.S.C.P.)
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Yelena G. Golubeva Ph.D., H.T. (A.S.C.P.)
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Scott M. Lawrence M.S., H.T. (A.S.C.P.) .

Editor information

Editors and Affiliations

  1. Center for Applied Proteomics and Molecular Medicine, George Mason University, Manassas, Virginia, USA

    Virginia Espina

Rights and permissions

Reprints and permissions

Copyright information

© 2017 Springer Science+Business Media LLC

About this protocol

Cite this protocol

Lawrence, S.M., Golubeva, Y.G. (2017). Optimization of Immunostaining for Prospective Image Analysis. In: Espina, V. (eds) Molecular Profiling. Methods in Molecular Biology, vol 1606. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6990-6_16

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-6990-6_16

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-6989-0

  • Online ISBN: 978-1-4939-6990-6

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation