Immunofluorescence

immunofluorescence

a technique used for light microscopy with a fluorescence microscope

antibodies to their target antigen

The technique of immunofluorescence uses the specificity of

The anitbodies are conjugated with a fluorescent tag to bind with specificity to a targeted biomolecule within a cell or cell surface

To allow visualization of the localization and distribution of the target molecule in a sample what must happen?

antibodies recognize and bind to a specific region of the antigen

What happens in primary immunofluorescence?

epitope

What is the specific region that an antibody binds to called?

The sample can be viewed under fluorescent microscope to confirm the presence of the antigen.

Once the antibody binds to the epitope in primary immunofluorescence, what can happen?

fluorophore to antibody

In the direct method there is a 1 to 1 stoichiometry of ____ to ____.

direct method

In which method of immunofluorescence can the fluorophore be directly conjugated to the primary antibody?

indirect method

In which method is there a secondary antibody, conjugated to a fluorophore, that binds specifically to the first untagged antibody, that can be used?

primary unlabeled antibody
signal detection and sensitivity

In the indirect method there is more than one fluoro labeled 2nd antibody that binds to a ____, thereby amplifying ___ and ____.

the primary antibody that is already bound to the antigen

The secondary antibody's binding sites in the indirect method are specific for what?

indirect

Which method allows for increased fluorescent signals during microscopy?

direct (primary) immunifluorescence

With method is less commonly used, but has notable advantages over the other method?

It reduces the number of steps in the procedure, savin time and reducing non-specific background signal.

the direct attachment of the messenger to the antibody in the primary method does what?

direct immunofluorescence

Which method is less sensitive and can result in false negatives?

direct immunofluorescence

Which method is much more expensive due to excessive use of primary antibodies?

Establish a range of concentration of antibodies to optimize specific interactions and minimize non-specific binding while providing a good signal.

What is the proper antibody dilution?

to block non-specific mAb binding to tissue proteins

Why use a blocking step?

heat and enzyme

What are some antigen retrieval methods?

Boiling FFPE tissue sections in buffer enhances immunoreactivity.

Why is heat a good retrieval method?

Enzyme digestion is not always effective and immunostaining may be patchy or absent which could result in tissue breakdown and false positive staining.

What is a disadvantage of enzyme retrieval method?

simpe step using only a single antibody
-cheaper
-shorter procedure

What are some advantages of direct immunofluorescent method?

only a 1:1 antigen to signal ratio
no antigen masking
fading of fluorescence signal
documentation of photography can be difficult
fluorescent microscope is required
mounting media requirements
difficult to standardize intensity of signal

What are some disadvantages of a direct immunofluorescent method?

Direct: uses a single antibody directed against the target of interest and the primary antibody is directly conjugated to a fluorophore.

Indirect: uses 2 antibodies and the primary id unconjugated while the secondary antibody is conjugated to the fluorophore. The secondary antibody is directed against the primary antibody for detection.

What is the difference between direct and indirect immunohistochemistry?

The fluorophore is directly conjugated to the primary antibody.

What is a direct immunohistochemical method?

indirect due to signal amplification courtesy of secondary antibodies

Which method is preferred?

multiple
a single

_____ secondary antibodies can bind to _____ primary antibody

It allows for signal amplification by increasing the number of fluorophore molecules per antigen.

What does multiple bindings to one primary antibody allow for?

Because a variety of different secondary antibodies and detection techniques can be used for a given primary antibody.

Why does the indirect method allow for more flexibility?

the photochemical alteration of a dye or a fluorophore molecule such that it permanently is unable to fluoresce.

What is photobleaching?

It is caused by cleaving of covalent bonds or non-specific reactions between the fluorophore and surrounding molecules which tend to be irreversible

How is photobleachign caused?

the molecules will eventually be destroyed by the light exposure necessary to stimulate them into fluorescing

How does photobleaching complicate the observation of fluorescent molecules?

protect the fluorophore from unnecessary ambient light exposure as well as minimize light exposure during evaluation of the specimen during microscopy

How can you prevent photobleaching?

by reducing or limiting the intensity or time-span of light exposure, by increasing the concentration of fluorophores, or by employing more robust fluorophores that are less prone to bleaching

How can loss of activity caused by photobleaching be controlled?

a family of fluorescent dyes invented by Molecular Probes

What are Alexa Fluor Dyes?

as cell and tissue labels in fluorescence microscopy and cell biology

Alexa Fluor dyes are frequently used as what?

to primary antibodies or to secondary antibodies to amplify signal and sensitivity or other biomolecules

Alexa Fluor dyes can be conjugated directly to what?

sulfonation and additional chemical modifications

The Alexa Fluor dyes were chemically synthesized through what?

it made them negatively charged and thus more hydrophilic and soluble than their parent dyes.

What did sulfonation do to the dyes?

pH-sensitive
photostable

The Alexa Fluor series dyes are less ______ and more ______ than the original dyes from which they were synthesized.

visible spectrum and extend into the infrared

The excitation and emission spectra of the Alexa Fluor series cover the

excitation maxima in nanometers

The individual members of the family are numbered according roughly to their

autofluorescence, extraneous undesired specific fluorescence, and nonspecific fluorescence.

Some problems that may arise from this immunofluorescence include

fluorescence emitted from the sample tissue or cell itself

Autofluorescence includes

a targeted antigen is impure and contains antigenic contaminants

extraneous undesired specific fluorescence occurs when

the loss of a probe's specificity due to fluorophore, from improper fixation, or from a dried-out specimen

Nonspecific fluorescence involves

this could result in either false positive or false negative signals due to non-specific binding

If proteins of interest become cross-linked this can cause what?

by attempting to quench tissue autofluorescence to improve signal-to-noise ratio

How can photobleaching be used in a favorable manner?

because antibodies do not penetrate the cell membrane when reacting with fluorescent labels

Why is immunofluorescence only limited to fixed cells when structures within the cell are to be visualized?

inside the cell

Antigenic material must be fixed firmly on the site of its natural localization is