Staining Techniques Histochemical Stains: involve chemical reactions Feulgen reaction -DNA Periodic Acid Shiff (PAS) -neutral and acidic polysaccharides - glycogen, mucous, basal laminae com.bioquantusers.org/products.php?page=ls&content=gall ery&sub=feulgen

Goblet cells PAS stain Intestinal Villus

Carbohydrate-rich Basal Laminae stain with PAS stain

Staining Techniques Localization (staining) of an enzyme AB + T AT + B ENZYME generate visible product provide substrate

Staining Techniques AB + T AT + B Acetylcholinesterase- neuromuscular junction ACETYL CHOLINESTERASE 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 laminin Antibodies 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

IMMUNOCYTOCHEMISTRY Direct 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 tissue Rinse with saline solution Incubate with conjugated antibody Rinse Mount on slide, view with microscope

DIRECT IMMUNOCYTOCHEMISTRY ADVANTAGES Specificity Less background staining DISADVANTAGES Low 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 tissue Rinse Incubate unlabeled primary antibody Rinse Incubate labeled secondary antibody Rinse Mount on slide, view with microscope

INDIRECT IMMUNOCYTOCHEMISTRY ADVANTAGES Amplification of the signal Can use labeled secondary with different primary antibodies DISADVANTAGES The nonspecific background may increase Takes longer to do Needs more reagents

LIMITATIONS OF IMMUNOCYTOCHEMISTRY Cross-reactivity Sensitivity Antigenicity - 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-spectrinrabbit anti-Goat-Rh donkey 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

In situ hybridization Labeled DNA or RNA probe Why?

In situ hybridization Labeled DNA or RNA probe Radioactive tag Digoxigenin Incubation with tissue Autoradiography 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 Univ. of Helsinki Fluorescence in situ hybridization of the all-human telomere probe, (T2AG3)n, to chromosome ends of the Pacific oyster (Crassostrea gigas).