staining techniques histochemical stains: involve chemical reactions feulgen reaction -dna
DESCRIPTION
Staining Techniques Histochemical 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. - PowerPoint PPT PresentationTRANSCRIPT
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
Goblet cells PAS stainIntestinal Villus
Carbohydrate-rich Basal Laminae stain with PAS stain
Staining Techniques
Localization (staining) of an enzyme
AB + T AT + BENZYME
generate visible product
provide substrate
Staining Techniques
AB + T AT + B
Acetylcholinesterase- neuromuscular junction
ACETYLCHOLINESTERASE
Other stains for ATPases, alkaline phosphatases, and others
A technique to localize specific molecules in an organ, tissue or cell.
IMMUNOCYTOCHEMISTRY
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……….
Polyclonal antibodies-A collection of distinct types of antibody molecules that
recognize the same antigen (antibodies A + B + C)
Monoclonal antibodies-A single type of antibody molecule that recognizes only
one epitope on an antigen (antibody A OR B OR C)
• Polyclonal antibodies•ADVANTAGES: recognize more epitopes in tissue•DISADVANTAGES: less specificity
• Monoclonal antibodies•ADVANTAGES: more specific•DISADVANTAGES: reduced signal possible
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?
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
IMMUNOCYTOCHEMISTRY Use of antibodies to detect specific
molecules (antigens) in a tissue
Antibody binds to an antigen in the tissue.
ANTIGEN
ANTIBODY
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
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.
INDIRECT IMMUNOCYTOCHEMISTRY– Primary antibody binds to the antigen.
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
INDIRECT IMMUNOCYTOCHEMISTRY
ADVANTAGESAmplification of the signalCan use labeled secondary with different
primary antibodies
DISADVANTAGESThe nonspecific background may increaseTakes longer to doNeeds more reagents
LIMITATIONS OF IMMUNOCYTOCHEMISTRY
Cross-reactivitySensitivityAntigenicity
-Frozen sections
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
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?
AUTORADIOGRAPHY
• Tissue with radiolabeled molecule
• Cover with photo emulsion• Radiation activates silver ->
silver grains• Develop and view
http://course1.winona.edu
In situ hybridizationLabeled DNA or RNA probe
Why?
In situ hybridizationLabeled DNA or RNA probe
Radioactive tagDigoxigenin
Incubation with tissueAutoradiography or Immunocytochemistry
www-bioc.rice.edu/bios576/immuno/immuno.html
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