CST Guide:

11
Section II: ANTIBODY APPLICATIONS
Immunofluorescence (IF)
Fluorescent analysis of cells and tissues
Immunofluorescence (IF) is a detection method that employs primary antibodies to reveal the localization
of target proteins and analyze their relative expression levels in cultured cells or tissue sections. Primary
antibodies can be directly labeled with a fluorescent tag (direct IF) or detected using a fluorescently
labeled secondary antibody (indirect IF). The ability to detect multiple fluorescent tags as distinct colors
within the same sample makes IF a powerful tool for investigating changes in subcellular localization,
because protein location can be analyzed in relation to other labeled organelle markers or other cellular
structures.
Evaluate fixation and permeabilization
protocols to achieve optimal staining.
Cell and tissue samples may be preserved using either crosslinking or denaturing fixatives. (Note: not all
samples are fixed for IF staining, such as in live cell staining.) The optimal fixation method is dependent
upon the target protein, its subcellular localization, and in some cases the antibody that is used. Intracellular
targets also require an additional permeabilization step. Detergents, such as Triton X-100 or Tween ®
20 work well for most antibodies but in some cases, methanol permeabilization may be necessary.
Optimizing fixation reagents can substantially improve staining results.
Formaldehyde
Methanol
chapter 11: Immunofluorescence (IF)
Methanol
permeabilization
improves intracellular
immunostaining for
some proteins.
Triton X-100
A number of factors can impact the ability
to achieve bright and specific IF staining.
• Antibody specificity and sensitivity • Target expression level • Species considerations
• Permeabilization agent used • Type and degree of fixation • Dye selection
• Antibody concentration and diluent • Cellular location of target
• Detection method (direct, indirect, amplified)
Keratin 8/18 (C51) Mouse mAb
#4546: IF analysis of HeLa cells,
fixed with formaldehyde (left) or
methanol (right), using #4546
(green). Red = Propidium Iodide
(PI)/RNase Staining Solution #4087.
Best with methanol fixation
Methanol
Nanog (D2A3) XP ® Rabbit mAb
(Mouse Specific) #8822: Confocal
IF analysis of F9 cells (Nanog positive)
(A) and NIH/3T3 cells (Nanog negative)
(B) was performed using #8822
and competitor antibodies at the
indicated concentrations.
IF Tips for Success
Use a well-characterized and specific
antibody for accurate IF results.
The primary antibody is an important reagent in successful IF experiments, because the specificity and
sensitivity of the antibody dictates the quality of experimental data.
Specificity of Nanog antibody is demonstrated by positive staining restricted
to the nucleus and by absence of staining in a Nanog-negative cell line.
A
B
CST #8822
(0.25 µg/ml)
Demonstrates specific nuclear
staining in Nanog-expressing cells
Crosslinking Fixatives
Examples: formaldehyde, formalin, glutaraldehyde, glyoxal
Note: Degree of crosslinking is dependent upon fixative
concentration, temperature, and time of fixation.
Advantages
• Excellent tissue morphology
AIF (D39D2) XP ® Rabbit mAb
#5318: IF analysis of HeLa cells,
fixed with formaldehyde (left) or
methanol (right), using #5318
(green). Red = Propidium Iodide
(PI)/RNase Staining Solution #4087.
Best with formaldehyde fixation
• Inactivate enzymes such as proteases and phosphatases
Limitations
• Toxicity risk to user
• Old formaldehyde solutions may induce autofluorescence
PDI Antibody #2446: Confocal IF analysis
of NIH/3T3 cells, permeabilized with
0.3% Triton X-100 (top) or methanol
(bottom), using #2446 (green) and
β-Actin (8H10D10) Mouse mAb #3700
(red). Blue pseudocolor = DRAQ5 ®
#4084 (fluorescent DNA dye).
Competitor antibodies used in accordance with manufacturer recommendations.
Competitor 1
(5.0 µg/ml)
Shows cytoplasmic and nuclear staining
in both positive and negative cell lines
Competitor 2
(2.0 µg/ml)
Demonstrates some staining in the
negative control and requires an
8-fold higher IgG concentration
Denaturing/Precipitating Fixatives
Examples: ethanol, methanol, acetone, acetic acid
Note: Denaturing fixatives may be combined with acetic
acid to enhance tissue penetration.
Advantages
• Loss of soluble proteins can allow presentation of antigens normally inaccessible to antibodies
• Work well with DNA stains
• Do not require further permeabilization
Limitations
• May induce cell swelling or lysis, if not used cold
• Loss of tertiary structure
• Not advisable for use with CD markers
• Protein loss may occur because of lack of crosslinking
Permeabilization Agents
For use after crosslinking fixatives, allows antibodies to penetrate cells and bind to intracellular antigens
Detergent Examples: Triton X-100, Tween ® , Saponin,
DOTMAC, SDS
Alcohol Examples: ethanol, methanol
Note: Alcohol permeabilization after fixation is not the
same as alcohol fixation.
• Typically not used with activation state-specific antibodies such as phospho-specific antibodies
Detergent
• Detergent permeabilization may require a separate step or may be included in blocking solution
and antibody diluent (depending on the detergent used).
Alcohol
• Alcohol permeabilization may be required for some proteins.
206 For Research Use Only. Not For Use in Diagnostic Procedures. See pages 302 & 303 for Pathway Diagrams, Application, and Reactivity keys.
www.cellsignal.com/cstif
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