Download - Staining Techniques Histochemical Stains: involve chemical reactions Feulgen reaction -DNA
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Staining TechniquesHistochemical Stains: involve chemical reactions
Feulgen reaction -DNA
Periodic Acid Shiff (PAS)-neutral and acidic polysaccharides- glycogen, mucous, basal laminae
http://bioquant-com.bioquantusers.org/products.php?page=ls&content=gallery&sub=feulgen
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Goblet cells PAS stainIntestinal Villus
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Carbohydrate-rich Basal Laminae stain with PAS stain
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Staining Techniques
Localization (staining) of an enzyme
AB + T AT + BENZYME
generate visible product
provide substrate
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Staining Techniques
AB + T AT + B
Acetylcholinesterase- neuromuscular junction
ACETYLCHOLINESTERASE
Other stains for ATPases, alkaline phosphatases, and others
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A technique to localize specific molecules in an organ, tissue or cell.
IMMUNOCYTOCHEMISTRY
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An organism creates antibodies to foreign molecules, ANTIGENS.
An antigen may have different regions, EPITOPES, that are recognized as foreign
by an organism.
First, a bit of immunology……….
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Polyclonal antibodies-A collection of distinct types of antibody molecules that
recognize the same antigen (antibodies A + B + C)
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Monoclonal antibodies-A single type of antibody molecule that recognizes only
one epitope on an antigen (antibody A OR B OR C)
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• Polyclonal antibodies•ADVANTAGES: recognize more epitopes in tissue•DISADVANTAGES: less specificity
• Monoclonal antibodies•ADVANTAGES: more specific•DISADVANTAGES: reduced signal possible
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EXPERIMENT:
- Homogenize a sample of human muscle containing a variety of cells (muscle cells, neurons, capillaries, connective tissue cells).
- Inject homogenate into a mouse.
WHAT HAPPENS IN THE MOUSE?
-Take of sample of mouse blood, extract the serum, stain a section of human muscle.
WHAT WILL BE STAINED IN THE HUMAN MUSCLE?
HOW DO WE GET STAINING OF ONLY MUSCLE MYOSIN?
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Representative myosin heavy chain (MHC) immunocytochemistry images of an emphysematous diaphragm after co-incubation with anti-laminin antibody and an antibody against one of the adult MHC isoforms.
Antibody against lamininAntibodies against different epitopes of myosin heavy chain
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IMMUNOCYTOCHEMISTRY Use of antibodies to detect specific
molecules (antigens) in a tissue
Antibody binds to an antigen in the tissue.
ANTIGEN
ANTIBODY
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IMMUNOCYTOCHEMISTRYDirect Immunocytochemistry: a visible marker is
directly attached to antibody binding the antigen
The antibody is conjugated to visible marker.•Fluorochrome•Enzyme (HRP)•Electron dense molecule (ferritin, gold)
Procedure: Fix the tissueRinse with saline solutionIncubate with conjugated antibodyRinseMount on slide, view with microscope
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DIRECT IMMUNOCYTOCHEMISTRYADVANTAGES
SpecificityLess background staining
DISADVANTAGESLow sensitivity if the antigen is present in the
tissue in low concentrations.Need to directly conjugate marker to antibody.
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INDIRECT IMMUNOCYTOCHEMISTRY– Primary antibody binds to the antigen.
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INDIRECT IMMUNOCYTOCHEMISTRY– Primary antibody binds to the antigen.– Secondary antibody binds to the primary antibody.– Secondary antibody is conjugated to a visible marker.
Procedure: Fix the tissueRinseIncubate unlabeled primary antibodyRinseIncubate labeled secondary antibodyRinseMount on slide, view with microscope
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INDIRECT IMMUNOCYTOCHEMISTRY
ADVANTAGESAmplification of the signalCan use labeled secondary with different
primary antibodies
DISADVANTAGESThe nonspecific background may increaseTakes longer to doNeeds more reagents
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LIMITATIONS OF IMMUNOCYTOCHEMISTRY
Cross-reactivitySensitivityAntigenicity
-Frozen sections
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Antibodies (immunoglobulins) of specific species are used as antigens to generate secondary antibodies.
ANTIGEN--> mouse antibody
Rabbit anti-mouse IgG
Goat anti-mouse IgG
Donkey anti-rabbit IgM
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QUESTION: Dr. Reist is studying the distribution of two proteins, FasII and spectrin in neurons. She would like to label both molecules in the same sample using double-labeling immunocytochemistry. She has these antibodies:
Primary antibodies: Secondary antibodies:rabbit anti-FasII mouse anti-rabbit-
FITC(fluorescein)mouse anti-FasII donkey anti-rabbit-FITC goat anti-FasII rat anti-mouse-Rh (rhodamine) rat anti-spectrin goat anti-mouse-Rh rabbit anti-spectrin rabbit anti-Goat-Rhdonkey anti-spectrin
What primary and secondary antibodies will successfully distinguish the distribution of FasII and spectrin in the same preparation?
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AUTORADIOGRAPHY
• Tissue with radiolabeled molecule
• Cover with photo emulsion• Radiation activates silver ->
silver grains• Develop and view
http://course1.winona.edu
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In situ hybridizationLabeled DNA or RNA probe
Why?
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In situ hybridizationLabeled DNA or RNA probe
Radioactive tagDigoxigenin
Incubation with tissueAutoradiography or Immunocytochemistry
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www-bioc.rice.edu/bios576/immuno/immuno.html
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Whole mount in situ hybridization views on E10.5 mouse embryos with Phox2a (A), En1 (B), Uncx4.1 (C) and Lmx1b (D) RNA-probes.Juha PartanenInstitute of Biotechnology, P.O.Box 56, FI-00014 Univ. of Helsinki
Fluorescence in situ hybridization of the all-human telomere probe, (T2AG3)n, to chromosome ends of the Pacific oyster (Crassostrea gigas).www.hsrl.rutgers.edu/mapping.html