Elements for

Immunohistochemi
stry
Immunocytochemis
try
Henry Li
Aug., 06, 2007
 Antibodies are immune system-related proteins called immunoglobulins. Each
antibody consists of four polypeptides– two heavy chains and two light chains
joined to form a "Y" shaped molecule.
 The amino acid sequence in the tips of the "Y" varies greatly among different
antibodies. This variable region, composed of 110-130 amino acids, give the
antibody its specificity for binding antigen. The variable region includes the ends
of the light and heavy chains. Treating the antibody with a protease can cleave
this region, producing Fab or fragment antigen binding that include the variable
ends of an antibody. Material used for the studies shown below originated from
Fab.
 The constant region determines the mechanism used to destroy antigen.
Antibodies are divided into five major classes, IgM, IgG, Iga, IgD, and IgE, based
on their constant region structure and immune function

http://commons.wikimedia.org
/wiki/Image:IgG_molecular_sur
face.jpg
Antibody produced by B
cells
Nature of antigen-antibody
interaction
Polyclonal Antibody
 Monoclonal Antibody

 Derived from a single clone and

specific for
 a single epitope

 1975- Kohler and Milstein developed

the
 hybridoma technique for
developing
 monoclonal antibodies
 Agglutination- reaction between antibody and
 particulate antigen

 Presence of excess antibody can inhibit

 agglutination (prozone effect)

 Each antibody “competes” for epitopes; if it

 binds one, it cannot link one antigen
 to another

 Some antibodies bind but do not agglutinate

 Epitope density or availability

General consideration of
antibody
 Behavior of monoclonal vs polyclonal antibodies

 Monoclonal antibodies tend to have high affinity

 Polyclonal antiserum will have mixture of low

 and high affinity antibodies

 Avidity vs affinity

 Antibodies can be cross-reactive (source of some

 autoimmune disorders)
 Research applications

 Structure-function analysis

 Recombinant antibodies

 “Humanized” antibodies
 Uses of antibodies in our
laboratory
 Western blotting (Immunoblotting)
 - Identification of protein antigen following SDS-PAGE

 Immunoprecipitation
 - Isolation of specific proteins + binding partners

 Immuno-histo-/cyto-chemistry
 - Localization of specific proteins in cells

 ELISA (Enzyme-Linked Immunosorbent Assay)

 - Detection of proteins in a sample

 Neutralization
 Deplete of endogenous proteins

 FACS: Fluorescence-Activated Cell Sorting

 For cell isolation
 Detection of specific
proteins:
SDS-PAGE and Western blot
 Separate proteins by
SDS PAGE
 Transfer proteins to
membranes (i.e.
Nitrocellulose)
 Block non-specific sites
on membrane
 Incubate with primary
antibody, wash
 Incubate with
secondary antibody,
wash
 Detect secondary
antibody
 Immunopreciptation:
Identification of protein-protein
 Steps:
interactions
 1. Attach antibody to beads via protein A
 2. Lyse cells to release antigen and its binding partners
 3. Mix cell lysate + antibody-coated beads (antibody binds
antigen)
 4. Purify antigen and its binding partners by centrifugation

bead

protein A

primary 
antibody
 Identification of protein-protein
interactions
by GST-pulldown assays
protein of interest

GST
add binding
partner

bead bead

glutathione wash

elute with glutathione

SDS­PAGE, Western blotting
 ELISA
(Enzyme-Linked Immunosorbent
Assay
 ELISA
(Enzyme-Linked Immunosorbent
Assay
FACS
 What is
Immunocyto/histochemistry
?
Using
antibodies to
localise
proteins
in cells and
tissues
 Immuno

 Cyto-/histo-

 Chemistry


Indirect vs Direct Antibody
Staining
 What is the difference between direct
and indirect immunostaining?
 Why do indirect Immunostaining?
 Avidin-Biotin
Immunohistochemistry
Avidin-Biotin Complex (ABC) Method
Labeled streptavidin-biotin (LSAB)
method
Immunofluorescence
Microscopy

Pituitary-derived sphere stained by S100+Nes

 Fluorescent antibody

 Antibodies can be labeled with fluorescent

dye

 Can localize binding sites on cells

 Dyes: Fluorescein, rhodamine,

phycoerythrin
 can be conjugated to Fc region of Ab
 (so antigen binding is unaffected)

 Absorb at one wavelength and emit at

another
 Keep in mind:
 Overall purpose of Immunocytochemistry:
 An antibody is used to link an antigen in a cell
with a stain or fluorophore that can more easily
be seen with a microscope than the original
antigen.
 Objects closer together than 200 –250 nm
can’t be resolved by fluorescence.
 Objects smaller than the limit of resolution
(200 nm) will be seen if sufficient antibody
binds
 Antibodies are critical (only as good as
their characterisation)
 Protocol
 Samples preparation & pre-treatment
 Blocking
 Primary antibodies
 Cocktails for double-/triple-staining (e.g.,
poly+monoclonal; ployclonal with diferent species:
sheep (goat)/Rabbit; monoclonal with different
isotypes: IgG1/IgG2a/IgG2b/IgM).
 Sequential staining : reduce the cross-reaction
 Incubation
 Secondary antibodies
 With/without DAPI (Hochest 33,342)
 Mounting & sealing
 Controls!!!!
 You want to know that you are
seeing what you think you are
seeing. Don’t You?
 Positive and Negative controls should
be performed or at least detailed.
 Alter only one variable at a time.
Recommended readings for
control
 Burry RW. 2000. Specificity Controls for
Immunocytochemical Methods. J Histochem
Cytochem 48(2):163-166.
 Clifford BS, Sawchenko PE. 2003. Magic peptides,
magic antibodies: Guidelines for appropriate
controls for immunohistochemistry. J Com Neurol
465(2):161-163.
 Holmseth, S., Lehre, K. P. and Danbolt, N. C.
(2006) Specificity controls for
immunocytochemistry. Anat Embryol (Berl), 211,
257-266
 Holmseth, S., Dehnes, Y., Bjornsen, L. P.,
Boulland, J. L., Furness, D. N., Bergles, D. and
Danbolt, N. C. (2005) Specificity of antibodies:
Unexpected cross-reactivity of antibodies directed
against the excitatory amino acid transporter 3
(EAAT3). Neuroscience, 136, 649-660
Available information:
IHC world:
www.ihcworld.com
 PROTOCOL ONLINE
http://www.protocol-
online.org/
 WIKIPEDIA
http://
en.wikipedia.org/wiki/Antibody
Handbook of Immunochemical Staining Methods,4th Editio
(Dako)

 Provided basic information and methodology necessary for

immunochemistry techniques. Contents include antibody, fixation, antigen
retrieval, staining methods, controls, background, in situ hybridization,
tissue processing, trouble shooting and glossary.
 http://www.dakousa.com/08002_25may06_ihcguidebook_lo.pdf
OMIM- Online Mendelian
Inheritance in ManTM
http://www.ncbi.nlm.nih.gov/sites/entrez?db=OMIM
 Wikipedia, the free
encyclopedia
http://en.wikipedia.org/wiki/Wikip
 Bookshelf
http://www.ncbi.nlm.nih.gov/sites/entrez?db=Book
 Video
 Double-labeling
 http://www.youtube.com/watch?v=GKYo1SmWjeQ
 Western Blot (Immunoblot)
 http://www.youtube.com/watch?v=k8NMRbgGozc&mode=related
&search=
 SDS-Polyacrylamide Gel Electrophoresis
 http://www.youtube.com/watch?v=8kkbxrbVlok&mode=rel
ated&search
 ELISA (Enzyme-Linked ImmunoabSorbant
Assay)
 http://www.youtube.com/watch?v=cJLQ4-g436g&mode=
related&search=