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 interactions Steps:
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
glutathione
bead
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
Incubation
Secondary 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
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.protocolonline.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=