Staining Techniques
Histochemical Stains: involve chemical reactions
Feulgen reaction
-DNA
http://bioquantcom.bioquantusers.org/products.php?page=ls&content=gall
ery&sub=feulgen
Periodic Acid Shiff (PAS)
-neutral and acidic polysaccharides
- glycogen, mucous, basal laminae
Intestinal Villus
Goblet cells
PAS stain
Carbohydrate-rich Basal Laminae stain with PAS stain
Staining Techniques
Localization (staining) of an enzyme
AB + T
ENZYME
AT + B
provide substrate
generate visible product
Staining Techniques
AB + T
ACETYL
CHOLINESTERASE
AT + B
Acetylcholinesterase- neuromuscular junction
Other stains for ATPases, alkaline phosphatases, and others
IMMUNOCYTOCHEMISTRY
A technique to localize specific molecules in an
organ, tissue or cell.
First, a bit of immunology……….
An organism creates antibodies to foreign
molecules, ANTIGENS.
An antigen may have different regions,
EPITOPES, that are recognized as foreign by
an organism.
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?
Antibody against laminin
Antibodies against different epitopes of myosin heavy chain
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.
IMMUNOCYTOCHEMISTRY
Use of antibodies to detect specific
molecules (antigens) in a tissue
Antibody binds to an antigen in the tissue.
ANTIBODY
ANTIGEN
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 doublelabeling 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-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
http://course1.winona.edu
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-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