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