Immunohistochemistry: Essential Elements and Beyond

91 Pages·2016·2.382 MB·English

by  Alexander E. Kalyuzhny (auth.)

Techniques in Life Science and Biomedicine for the Non-Expert
Series Editor: Alexander E. Kalyuzhny
Alexander E. Kalyuzhny
Immuno-
histochemistry
Essential Elements and Beyond
Techniques in Life Science and Biomedicine
for the Non-Expert
Series editor
Alexander E. Kalyuzhny, University of Minnesota, Minneapolis, MN, USA
The goal of this series is to provide concise but thorough introductory guides to
various scientific techniques, aimed at both the non-expert researcher and novice
scientist. Each book will highlight the advantages and limitations of the technique
being covered, identify the experiments to which the technique is best suited, and
include numerous figures to help better illustrate and explain the technique to the
reader. Currently, there is an abundance of books and journals offering various
scientific techniques to experts, but these resources, written in technical scientific
jargon, can be difficult for the non-expert, whether an experienced scientist from a
differentdisciplineoranewresearcher,tounderstandandfollow.Thesetechniques,
however, may in fact be quite useful to the non-expert due to the interdisciplinary
natureofnumerousdisciplines,andthelackofsufficientcomprehensibleguidesto
such techniques can and does slow down research and lead to employing
inadequate techniques, resulting in inaccurate data. This series sets out to fill the
gap in this much needed scientific resource.
More information about this series at http://www.springer.com/series/13601
Alexander E. Kalyuzhny
Immunohistochemistry
Essential Elements and Beyond
123
Alexander E. Kalyuzhny
University of Minnesota
Minneapolis, MN
USA
ISSN 2367-1114 ISSN 2367-1122 (electronic)
Techniques inLife ScienceandBiomedicine forthe Non-Expert
ISBN978-3-319-30891-3 ISBN978-3-319-30893-7 (eBook)
DOI 10.1007/978-3-319-30893-7
LibraryofCongressControlNumber:2016933323
©SpringerInternationalPublishingSwitzerland2016
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To Dora and Henry
Preface
Thisvolumechampionsanewbookseries,ofwhichIamhonoredtobetheeditor
and to have the opportunity to be the first contributor. As with any endeavor, it is
bothexcitingandfrighteningtobethefirst.Whatiftheformatofthebookisnotan
optimal match for the presented subject? What if the illustrations are not clear and
aredifficulttodigest?Whatifitistoolong,andafterreadingthefirstfewpagesthe
reader feels bored and the book finds its place on a dusty shelf among outdated
manuals and last century’s catalogs? Fortunately, we managed to convert all
of these “what if” worries into a concise writing plan that serves as a road map to
help us reach our goal: showing the elements of immunohistochemistry and
explaining how they come together to make this technique work.
IwishtoacknowledgeMartinWessendorf,whomadethebiggestimpactonmy
career development by introducing me to the exciting world of immunohisto-
chemistry more than twenty years ago.
Martin remains my mentor and good friend, and this book could not have been
written without his encouragement and support.
Finally, my special and sincere thanks go to Jennifer Kalyuzhny for her out-
standing editorial assistance.
vii
Contents
1 Short Historic Overview. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
2 Primary Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
2.1 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
2.2 Quiz. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
3 Choosing Commercial Suppliers of Primary Antibodies. . . . . . . . . . 11
3.1 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
3.2 Quiz. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
4 Detection and Visualization of Antigens . . . . . . . . . . . . . . . . . . . . . 15
4.1 Secondary Antibodies. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
4.2 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
4.3 Quiz. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
4.4 How to Choose Secondary Antibodies for IHC? . . . . . . . . . . . . 19
4.5 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
4.6 Quiz. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
4.7 Fluorescent Probes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
4.8 Enzyme Conjugates and Their Chromogenic Substrates . . . . . . . 25
4.9 Advantages and Disadvantages of Fluorescence
and Enzymatic Chromogenic Detection. . . . . . . . . . . . . . . . . . . 26
4.10 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
4.11 Quiz. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
5 Ancillary Reagents for IHC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
5.1 IHC Buffers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
5.2 Mounting Media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
5.3 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
5.4 Quiz. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
ix
x Contents
6 Preparing Tissues for IHC Staining . . . . . . . . . . . . . . . . . . . . . . . . 37
6.1 Tissue Fixation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
6.1.1 Cross-Linking Fixatives . . . . . . . . . . . . . . . . . . . . . . . . 38
6.1.2 Precipitating Fixatives . . . . . . . . . . . . . . . . . . . . . . . . . 39
6.1.3 Perfusion Fixation . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
6.1.4 Immersion Fixation . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
6.2 Cutting Tissue Sections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
6.2.1 Frozen Tissue Sections. . . . . . . . . . . . . . . . . . . . . . . . . 43
6.2.2 Paraffin-Embedded Tissue Sections . . . . . . . . . . . . . . . . 45
6.3 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
6.4 Quiz. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
7 Staining Protocols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
7.1 Fluorescence Protocols. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
7.1.1 Direct Fluorescence Detection. . . . . . . . . . . . . . . . . . . . 50
7.1.2 Indirect Fluorescent Detection. . . . . . . . . . . . . . . . . . . . 51
7.1.3 Multi-Color Fluorescence Detection. . . . . . . . . . . . . . . . 51
7.1.4 Fluorescent Counterstaining . . . . . . . . . . . . . . . . . . . . . 52
7.2 Chromogenic Protocols. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
7.2.1 LSAB Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
7.2.2 ABC Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
7.2.3 Non-Biotin-Based Detection . . . . . . . . . . . . . . . . . . . . . 55
7.2.4 Counterstaining in Chromogenic Detection. . . . . . . . . . . 55
7.3 IHC Controls. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56
7.3.1 Tissue Antigen Controls. . . . . . . . . . . . . . . . . . . . . . . . 56
7.3.2 Endogenous Tissue Condition Controls . . . . . . . . . . . . . 57
7.3.3 Reagents Controls . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
7.4 Selection and Optimizaion of Primary Antibodies . . . . . . . . . . . 59
7.5 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
7.6 Quiz. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
8 Microscopy Examination and Collection of Digital Images
and Their Presentation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
8.1 Operating a Bright-Field Microscope and Collecting IHC
Images . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
8.2 Operating Fluorescence Microscope and Collecting
IHC Images. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68
8.3 Presenting Immunohistochemistry Data. . . . . . . . . . . . . . . . . . . 70
8.4 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73
8.5 Quiz. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73
Contents xi
9 Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75
Quiz Answer Keys . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83