Immunolocalization for EMImmunolocalization for EM

Using immunoglobulin molecules as tags for select proteins and carbohydrates.

Visualized by using colloidal gold or enzyme reactions

Leishmania megasome labeled with 10nm gold

Immune ResponsesImmune Responses

1. Humoral Humoral: B lymphocytes produce antibodies recognizing an antigen from foreign substance. Antibodies are then secreted into blood stream.

Immune ResponsesImmune Responses

1. Humoral Humoral: B lymphocytes produce antibodies recognizing an antigen from foreign substance. Antibodies are then secreted into blood stream.

2. Cell-mediated:Cell-mediated:Mature T lymphocytes - antigen responding, response control, and response mediating cells

IgG

Immunoglobins (Ig)

IgA

IgG

Immunoglobins (Ig)

IgA IgM

IgG

Immunoglobins (Ig)

GlossaryGlossary

AntibodyAntibody (anti-foreign body) is a protein produced by a white cell (B lymphocyte).

GlossaryGlossary

AntibodyAntibody (anti-foreign body) is a protein produced by a white cell (B lymphocyte).

AntigenAntigen (antibody generating substance) is any agent, such as a chemical or microorganism that is recognized by the antibody. Not all antigens are immunogens (e.g hapten).

GlossaryGlossary

AntibodyAntibody (anti-foreign body) is a protein produced by a white cell (B lymphocyte).

AntigenAntigen (antibody generating substance) is any agent, such as a chemical or microorganism that is recognized by the antibody. Not all antigens are immunogens (e.g hapten).

Immunogen :Immunogen : Any substance to which an animal responds by making antibodies. All immunogens are antigens.

GlossaryGlossary

AntibodyAntibody (anti-foreign body) is a protein produced by a white cell (B lymphocyte).

AntigenAntigen (antibody generating substance) is any agent, such as a chemical or microorganism that is recognized by the antibody. Not all antigens are immunogens (e.g hapten).

Immunogen :Immunogen : Any substance to which an animal responds by making antibodies. All immunogens are antigens.

Antigen binding siteAntigen binding site - relatively small region of an antibody that binds to the antigen.

EpitopeEpitope (antigenic determinant) - is that part of an antigen that is recognized by a single antibody.

EpitopeEpitope (antigenic determinant) - is that part of an antigen that is recognized by a single antibody.

HaptenHapten - low molecular weight compounds (such as plant hormones) that typically do not elicit a spontaneous immune response but can be recognized by antibodies. Typically attached to an immunogen.

EpitopeEpitope (antigenic determinant) - is that part of an antigen that is recognized by a single antibody.

HaptenHapten - low molecular weight compounds (such as plant hormones) that typically do not elicit a spontaneous immune response but can be recognized by antibodies. Typically attached to an immunogen.

HybridomaHybridoma - fusion product between B cell and myeloma cell (“immortal cell”).

EpitopeEpitope (antigenic determinant) - is that part of an antigen that is recognized by a single antibody.

HaptenHapten - low molecular weight compounds (such as plant hormones) that typically do not elicit a spontaneous immune response but can be recognized by antibodies. Typically attached to an immunogen.

HybridomaHybridoma - fusion product between B cell and myeloma cell (“immortal cell”).

HAT selectionHAT selection - culture media that contains hypoxanthine, aminopterin and thymadine. A selective media that only allows hybridomas to grow.

Terms used in ImmunolabelingTerms used in Immunolabeling

Primary antibody:Primary antibody: An antibody that is specific to the antigen of the sample to be localized. Can be conjugated to a signal (gold, fluorochrome or enzyme).

Terms used in ImmunolabelingTerms used in Immunolabeling

Primary antibody:Primary antibody: An antibody that is specific to the antigen of the sample to be localized. Can be conjugated to a signal (gold, fluorochrome or enzyme).

Secondary antibody:Secondary antibody: An antibody that recognizes a primary antibody. Usually always is conjugated to signal.

Terms used in ImmunolabelingTerms used in Immunolabeling

Primary antibody:Primary antibody: An antibody that is specific to the antigen of the sample to be localized. Can be conjugated to a signal (gold, fluorochrome or enzyme).

Secondary antibody:Secondary antibody: An antibody that recognizes a primary antibody. Usually always is conjugated to signal.

DiluentDiluent: Physiologic buffer and non-specific protein (e.g. albumin or non-fat milk) used in diluting the antibodies. Sometimes detergent added to decrease surface tension of sections.

Block:Block: Physiologic buffer, high salt, and non-specific protein. The protein adheres to any “sticky” sites that might allow non-specific binding of antibodies.

Block:Block: Physiologic buffer, high salt, and non-specific protein. The protein adheres to any “sticky” sites that might allow non-specific binding of antibodies.

Etching:Etching: treating resin sections with HCl or sodium borohydride to reduce steric hindrance or expose hidden antigenic sites.

Polyclonal:Polyclonal:Antibodies are collected from sera of exposed animal, - or -a combination of monoclonal coloniesmonoclonal colonies is combined.

Antibody ProductionAntibody Production

Polyclonal:Polyclonal:Antibodies are collected from sera of exposed animal, - or -a combination of monoclonal coloniesmonoclonal colonies is combined.

Can be any animal: Rabbit, Goat, Horse, Rabbit, Goat, Horse, Rat, SheepRat, Sheep, etc…

Antibody ProductionAntibody Production

Polyclonal:Polyclonal:Antibodies are collected from sera of exposed animal, - or -a combination of monoclonal coloniesmonoclonal colonies is combined.

Can be any animal: Rabbit, Goat, Horse, Rabbit, Goat, Horse, Rat, SheepRat, Sheep, etc…

Suite of antibodies recognizing multiple antigenic sites of injected biochemical.

Antibody ProductionAntibody Production

Monoclonal:Monoclonal: Individual B lymphocyte hybridoma is cloned and cultured. Secreted antibodies are collected from culture media.

Monoclonal:Monoclonal: Individual B lymphocyte hybridoma is cloned and cultured. Secreted antibodies are collected from culture media.

Typically BALBc miceBALBc miceSometimes Rat Rat (ascites fluid)..

Monoclonal:Monoclonal: Individual B lymphocyte hybridoma is cloned and cultured. Secreted antibodies are collected from culture media.

Typically BALBc miceBALBc miceSometimes Rat Rat (ascites fluid)..

Antibodies recognize one antigenic binding site of the antigen.

Determining which B cell to

clone

ELISAor

Immuno-fluorescence

Generic TEM Immunolabeling ProtocolGeneric TEM Immunolabeling Protocol

Fixation:

Glutaraldehyde only

Dehydration

Embedding in methacrylate resin (e.g. LR White, Lowicryl, or Quetol)

Section

ImmunolabelImmunolabel

Optional - Post-stain

ControlsControls

Adsorption - primary antibody exposed to excess of antigen to remove any labeling.

Label without antibody - no antibody, shows labeling not due to reaction with label

Omission of primary antibody - the secondary or tertiary antibodies should not recognize the tissue.

Pre-immune sera - collected from animals that have not produced antibodies, or use “normal serum” from non-immunized animals of same species

Immunolabeling SectionsImmunolabeling Sections

- Float grids on blocking solution- Incubate in primary antibody

- Wash thoroughly with buffer to remove unbound antibody

Incubate with secondary antibody

or protein A/G

Wash again to remove unbound secondaryLast wash should be water.

Double LabelingDouble Labeling

Incubate sections with second primary antibody of interest

Incubate with secondary - different size gold

Note steric hinderence at asterisk

10 nm and 5 nm gold

Microtubules labeled with anti-beta tubulin

How are the antibodies coupled to the gold?The proteins are attached to the gold particles by:

a). Charge attraction (Lysine)b). Hydrophobic attraction (Tryptophan) andc). Sulfur binding (Cysteine and Methionine)

Strongest binding suggested to be the sulfur "hinge" joining the the two Fc regions

Using Protein A or Protein G instead of secondary antibody

Ferritin enhanced labeling Silver enhanced

Freeze fractureimmunolabeled

Freeze fractureimmunolabeled

Negative stainImmunolabeled

Etching to expose antigenic sitesEtching to expose antigenic sites