Antibodies and
immunofluorescence labeling
http://www.cellsignal.com/common/content/content.jsp?id=apps-immunofluorescence
What is an antibody?
Antibodies, or immunoglobulins, are glycoproteins
composed of 2 heavy and 2 light chains.
Both light and heavy chains have different variable
regions depending how the DNA is arranged and can
form more than 15,000,000 combinations
“Somatic
Recombination” is
the mechanism by
which some DNA
is removed from
the antibody
coding region
Variable regions
http://www.biology.arizona.edu/immunology/tutorials/immunology/05t.html
How do you create a specific
antibody?
1. Immunize mouse to desired
antigen
2. Isolate B cells (mortal) from
spleen
3. Cultivate myeloma cells
(immortal)
4. Fuse both cell types
5. Separate individual cells and
cultivate
6. Screen for antibody production
7. Multiply and harvest successful
hybridoma cells
http://en.wikipedia.org/wiki/Hybridoma_technology
Different immunolabeling
techniques
• Immunofluorescence straining: antibodies are
conjugated with a fluorescent dye that can be
excited to emit detectable light
• Immunoenzymological staining: antibodies are
conjugated with an enzyme that catalyzes the
transitions of an added substrate into an insoluble
particle that can be localized
• Immunocolloidal gold staining: antibody is
conjugated with gold that is used as a marker
QUIZ 1!
1. Why can different antibodies bind to so many
different biomolecules?
2. Why do you think the B cells are fused with
myeloma cells?
3. What is the main difference between
immunofluorescence staining, immunoenzymological
staining, and immunocolloidal gold staining?
Function of immunofluorescence
Antibodies target
specific biomolecules
(antigens) and reveal
their distribution and
density throughout the
sample via conjugated
fluorophore
http://alizeepathology.com/samples/1
Green = AlexaFluor 488nm dye conjugated to actin; Blue = DAPI; Red = auto-fluorescence
Primary/Direct
immunofluorescence
• Uses a single type of ‘antibody
• The ‘primary’ antibody binds directly to target
biomolecule (antigen) and fluoresces via the
attached fluorophore
http://www.abcam.com/secondary-antibodies/direct-vs-indirect-immunofluorescence
Secondary/Indirect
immunofluorescence
• Uses 2 antibody types
• The ‘primary’ antibody binds directly to the target
biomelocule (antigen) but has no attached
fluorophore
• The ‘secondary’ antibody binds to the ‘primary’
antibody and fluoresces via the attached fluorophore
http://www.abcam.com/secondary-antibodies/direct-vs-indirect-immunofluorescence
Example of primary/direct
immunofluorescence
SLBP (stem-loop
binding protein)
antibody binding to
hairpin loops on
mRNA in Hep G2
cells (liver tissue
with hepatocellular
carcinoma)
http://www.scbt.com/datasheet-362617.html
Example of secondary/indirect
immunofluorescence
Smooth muscle
antibody binding to
actin, vimentin,
desmin, and tubulin
(all microfilaments)
in Hep G2 cells
(liver tissue with
hepatocellular
carcinoma)
http://www.birmingham.ac.uk/facilities/clinical-immunologyservices/autoimmunity/liver-associated-antibodies/Liver-antibodies.aspx
Primary versus Secondary
immunofluorescence
TIME
Primary:
Less steps require less
time
Secondary:
Additional steps require
additional time.
Primary versus Secondary
immunofluorescence
COMPLEXITY
Primary:
Less steps in the
protocol makes the
experiment less
complex
Secondary:
Additional steps,
selection of secondary
antibodies, and
potential background
interactions add
additional complexity
Primary versus Secondary
immunofluorescence
COST
Primary:
Secondary:
Primary antibodies with
an attached fluorophore
(conjugated) are
typically more expensive
than unconjugated
primary antibodies
Secondary antibodies
are less expensive and
can used against
different primary
antibodies
Primary versus Secondary
immunofluorescence
SENSITIVITY
Primary:
Less conjugated
antibodies bind overall,
so the signal is less
intense
Secondary:
Several secondary
antibodies bind to the
primary antibody and
produce an amplified
signal
Primary versus Secondary
immunofluorescence
FLEXIBILITY
Primary:
Pre-conjugated primary
antibodies have limited
flexibility
Secondary:
Ability to use different
secondary antibodies
increase flexibility of
experiment
Primary versus Secondary
immunofluorescence
SUMMARY!
Primary:
•
•
•
•
•
Less time required
Less complexity
Higher cost
Less sensitivity
Less flexibility
Secondary:
•
•
•
•
•
Higher time required
Higher complexity
Less cost
Higher sensitivity
Higher flexibility
QUIZ 2!
1. Is primary or secondary immunofluorescence
more complex?
2. If you were attempting to detect a biomolecule
that likely has very low concentration in the
sample, would you choose to use secondary or
primary immunofluorescence?
Immunofluorescence protocol for
staining cell cultures
General Procedure
1. Coat coverslips in polyethyleneimine (for
increased adhesion)
2. Rinse coverslips with water
3. Dry coverslips
4. Grow cells on glass coverslips
5. Rinse with Phosphate-based saline (PBS)
Immunofluorescence protocol for
staining cell cultures
Fixation
1. Either Incubate cells with 100% methanol or 4%
(para)formaldehyde in PBS
2. Wash cells three times with cold PBS
Immunofluorescence protocol for
staining cell cultures
(Optional) Permeabilization
1. Incubate samples with PBS and 0.1-0.25% Triton
X-100 (a detergent that improve antibody
penetration)
2. Wash cells with PBS three times
Immunofluorescence protocol for
staining cell cultures
Blocking and incubation
1. Incubate cells with
-1% BSA (bovine serum albumin)
-22.52mg/ml glycine (reacts with any excess
formadehyde) in PBS + tween 20 (a surfactant) for 30
minutes
2. Incubate cells with primary antibodies in 1% BSA
in PBS + tween 20 for 1 hour at room temperature
Immunofluorescence protocol for
staining cell cultures
Blocking and incubation
3. Wash cells three times in PBS
4. Incubate cells with secondary antibody in 1% BSA
for 1 hour at room temperature in the dark
5. Wash antibody solution three times with PBS in the
dark
Immunofluorescence protocol for
staining cell cultures
Mounting
1. Mount coverslip with a drop of mounting medium
2. Seal coverslip
3. Store in darkness
LAST QUIZ!
1. If you were examining a human cell culture with
indirect immunofluorescence using bovine primary
antibodies and rabbit secondary antibodies, what
type of albumin serum would you use when
blocking and incubating the cell culture?
General references
1. http://www.bio.davidson.edu/genomics/method/IMF.html
2. http://www.abcam.com/secondary-antibodies/direct-vsindirect-immunofluorescence
3. http://www.microscopemaster.com/immunofluorescencemicroscopy.html
4. http://en.wikipedia.org/wiki/Antibody
5. http://atlasgeneticsoncology.org/Genes/CEACAM1ID40044
ch19q13.html
6. http://www.biology.arizona.edu/immunology/tutorials/immun
ology/05t.html
7. http://rockland-inc.com/BasePage.aspx?id=41002
8. http://www.piercenet.com/method/overview-antibodyproduction-purification
9. http://www.scbt.com/datasheet-362617.html
Academic References
1. Agrawal A, Godar DE. Simultaneous Detection and
Semiquantification of DNA Damage in Normal and Apoptotic Cells:
Triple-Immunofluorescent Labeling Using DAPI, Antibodies, and TUNEL.
Applied Immunohistochemistry & Molecular Morphology. 2012;20:402409.
2. Bordeaux J, Welsh AW, Agarwal S, et al. Antibody validation.
BioTechniques. 2010;48:197-209.
3. Galluzzi L, Aaronson SA, Abrams J, et al. Guidelines for the use and
interpretation of assays for monitoring cell death in higher eukaryotes.
Cell Death Differ. 2009;16:1093-1107.
4. Zinchuk V, Zinchuk O, Okada T. Quantitative colocalization analysis of
multicolor confocal immunofluorescence microscopy images: Pushing
pixels to explore biological phenomena. Acta Histochem Cytochem.
2007;40:101-111.
Indirect fluorescence picture
example
Skin and vaginal tissues
exposed to UV damage at
time of damage (a,b), 1
hour post-damage (c,d),
and 24 hours post damage
(c,f)
DAPI = blue (nuclei)
TUNEL = green (apoptosis)
CPD (cyclobutane pyrimidine dimers)
antibody = red (DNA damage)
Agrawal A, Godar DE. Simultaneous Detection and Semiquantification of DNA Damage in Normal and Apoptotic Cells: Triple-Immunofluorescent Labeling
Using DAPI, Antibodies, and TUNEL. Applied Immunohistochemistry & Molecular Morphology. 2012;20:402-409