CST Guide:

Section II: ANTIBODY APPLICATIONS
chapter 11: Immunofluorescence (IF)
Perform a titration to determine
optimal antibody concentration.
It is important to use an antibody at its optimal concentration. Using a lower concentration (more
dilute) will diminish positive signal, but just as importantly, using higher concentration (less dilute) will
increase background and decrease the signal-to-noise ratio. CST scientists routinely perform titrations
using positive and negative cell lines to identify the concentration that gives optimal signal with minimal
background staining. This recommended antibody dilution information is determined and provided with
every lot of an IF-validated antibody.
Antibody titration curves determine optimal concentration
for maximal signal-to-noise in IF results.
A
Mean Fluorescence Intensity
1200
1000
800
600
400
200
Optimal concentration for highest
signal-to-noise with minimal background.
50.0
45.0
40.0
35.0
30.0
25.0
20.0
15.0
10.0
5.0
Signal-to-Noise
Choose a detection method that suits your
target protein abundance and sample type.
Conjugated primary antibodies (direct detection) offer convenience and a shorter protocol. Secondary
antibodies conjugated to fluorochromes (indirect detection) may provide stronger signal intensity
because multiple secondary antibodies can bind each primary antibody, but require using primary
antibodies from different species when examining multiple targets within the same sample. Depending
on the target expression level and sample type, direct and indirect IF may still be too dim for some
assays, necessitating further amplification, such as avidin/biotin or tyramide. Avidin/biotin can improve
signal intensity, but it may not be possible to completely block all nonspecific signal from endogenous
biotin. More dramatic signal amplification can be achieved using tyramide amplification, which utilizes
HRP-conjugated secondary antibodies to catalyze the deposition of fluorochrome-conjugated tyramide
around the target. Tyramide amplification is very useful when performing IF in formalin-fixed paraffinembedded
(FFPE) tissues. Signal amplification helps distinguish true signal from autofluorescence and
negates issues related to antigen quality or scarcity, from either unmasking or protein loss. Tyramide
also enables multiplex staining with antibodies from the same species (1).
Retina stained with two directly conjugated
antibodies from the same species
Synapsin-1 (D12G5) XP ® Rabbit mAb (Alexa Fluor ® 594 Conjugate) #13556:
Confocal IF analysis of rat retina using #13556 (red) and Neurofilament-L (C28E10)
Rabbit mAb (Alexa Fluor ® 488 Conjugate) #8024 (green). Blue pseudocolor =
DRAQ5 ® #4084 (fluorescent DNA dye).
0.25 0.5 0.75 1 1.25 1.5 1.75 2 2.25
Concentration (µg/ml)
MFI (+) MFI (–) Signal-to-Noise
B
Dye-conjugated
secondary antibodies
can improve signal
intensity for low
abundance protein
targets.
Neurofilament-L (C28E10) Rabbit
mAb #2837: Confocal IF analysis of
neuroepithelial clusters differentiated
from human iPS cells, using #2837 detected
with Anti-rabbit IgG (H+L), F(ab’) 2
Fragment (Alexa Fluor ® 555 Conjugate)
#4413 (blue) and β3-Tubulin (TU-20)
Mouse mAb #4466 detected with
Anti-mouse IgG (H+L), F(ab’) 2 Fragment
(Alexa Fluor ® 488 Conjugate) #4408
(red). Blue pseudocolor = DRAQ5 ®
#4084 (fluorescent DNA dye).
C
Amplification enables detection of low abundance
protein targets in paraffin-embedded tissue.
0.031 µg/ml 0.063 µg/ml 0.125 µg/ml 0.25 µg/ml 1 µg/ml
MUC1 (D9O8K) XP ® Rabbit mAb #14161: Graph depicting Mean Fluorescence Intensity (MFI) of ZR-75 cells (MUC1 expressing) and
HCT 116 cells (MUC1 negative) using #14161, and calculated signal-to-noise (A). IF analysis of ZR-75 cells (B) and HCT 116 cells (C) at
varying concentrations, as indicated, using #14161. Red = Propidium Iodide (PI)/RNase Staining Solution #4087.
A
B
C
E-Cadherin (24E10) Rabbit mAb
#3195: IF analysis of FFPE human
metastatic lymph node using #3195
detected with a conjugated secondary
antibody (A,B) or detection with Antirabbit
IgG, HRP-linked Antibody #7074
and a FITC-tyramide conjugate (C).
Tissue Autofluorescence
Low Exposure
High Exposure
Low Exposure
Organelle Marker Samplers
Organelle Marker Samplers provide a convenient collection of primary antibodies targeting well-established
organelle associated proteins. For the most up-to-date listing of Organelle Marker Samplers, please go to
www.cellsignal.com/organelles
References:
1. Toth, Z.E. and Mezey, E. (2007) J. Histochem. Cytochem. 55, 545–554.
208 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
209