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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=