Dr Rania Abo-Shady
ASS.Prof. of Clinical Pathology
Ain Shams University
• Immunofluorescence is a technique allowing the
visualization of a specific protein or antigen in tissue
sections by binding a specific antibody chemically
conjugated with a fluorescent dye such as fluorescein
• The specific antibodies are labeled with a compound
(FITC) that makes them glow an apple-green color
when observed microscopically under ultraviolet
• Fluorescence is the property of certain molecules
or fluorophores to absorb light at one wave length
and emit light at longer wave length (emission
wavelength) when it is illuminated by light of
a different wavelength (excitation wavelength).
• The incident light excites the molecule to a higher
level of vibrational energy. As the molecules return
to the ground state, the excited fluorophore emits
a photon(= fluorescence emission ).
There are two major types of
immunofluorescence staining methods:
• 1) direct immunofluorescence: staining in
which the primary antibody is labeled with
• 2) indirect immunofluorescence: staining in
which a secondary antibody labeled with
fluorochrome is used to recognize a primary
Advantages of indirect:
(1). Gives an amplification effect -- more tag or label ('signal')
per molecule of target protein.
(2). Requires only one labeled antibody to identify many
proteins. Same labeled secondary antibody can be used to bind
to ("light up") many different proteins. (Preparation of labeled
antibody is difficult and expensive.)
(a). A different primary antibody is used for each target protein.
(Not labeled -- no tag.) Variable part of primary antibody binds
to specific part of target protein.
(b). The secondary antibody binds to the constant part of the
primary antibody. Therefore a sample of the same (labeled or
tagged) batch of secondary antibody can bind to many different
(unlabeled) primary antibodies
• Indirect immunofluorescence uses two
antibodies; the first (the primary antibody)
recognises the target molecule and binds to it,
and the second (the secondary antibody),
which carries the fluorophore, recognises the
primary antibody and binds to it.
• For the determation of autoantibodies, tissue
sections are used as antigen substrates.
• If the sample is positive, specific antibodies in
the diluted serum sample attach to the
antigens coupled to a solid phase.
• In a second step, the attached antibodies are
stained with fluorescein-labelled anti-human
antibodies and visualized with the
Indirect immunofluorescence assay:
A laboratory test used to detect antibodies in serum
or other body fluid.
Examples of autoantibodies:
– Anti-dsDNA Abs.
– ANA .
– Anti LKM.
– Antithyroid Abs.
Indirect immunofluorescence is considered the
standard technique for detection of autoantibodies.
It offers unique advantages:
• A negative result excludes the presence of all these
• For every antibody there is a characteristic
• High specificity through visual discrimination:
Antibodies are localized morphologically in exactly
the same spots as their corresponding antigens.
• The combination of different substrates in one test
field is highly suitable for determining autoantibody
profiles (mouse –stomach –kidney substrate CT3 )
Autoantibodies are detected on
– Anti-dsDNA Ab Crithedia Lucilae substrate
– ANA on Hep-2 substate
on mouse stomach kidney substrate
– ASMA. on mouse stomach kidney substrate(CT2)
– Anti LKM on mouse liver stomach kidney (CT3)
– ANCA on neutrophil substrate
– Antithyroid Abs on Thyroid tissue
Advantage of Hep2 cells over rodent tissue
i. Higher sensitivity (greater Ag expression)
ii. Human origin ensure better specificity
iii. Cell division rates are higher so cell cycle
dependent Ab are easily identified
iv. Nucleus are much larger ,visible & complex
nucleolar detail can be seen
v. Ags distribution are uniform not obscuring
Antinuclear antibodies (ANA)
• Are autoantibodies directed against various nuclear antigens,
and are used to report the titer of the ANA and the pattern of
nuclear staining of the ANA.
• Comment on :
-Type of substrate
• STAINING PATTERNS
• Diffuse / homogeneous: antibodies to histone
• Rim: antibodies to nuclear envelope proteins
and to double-stranded (ds) DNA
• Speckled: antibodies to Sm, RNP, Ro/SS-A,
La/SS-B, and other antigens
• Nucleolar: associated with diffuse scleroderma
• Centromeric: highly specific for the CREST
Anti-LKM on mouse liver stomach
• The pattern was consistently found to be that of a
typical LKM antibody without any evidence of a
mitochondrial antibody pattern is as follows;
Liver – strong positive cytoplasmic stain
Kidney – strong positive cytoplasmic stain in inner
proximal tubules, negative distal tubules.
Stomach – negative