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IHC Tip 4: Which counterstains are most compatible with my antibody staining?

Bio-Rad
May 19, 2015

Like controls, counterstaining is crucial for every IHC experiment, as the counterstain provides background contrast and puts the observed staining into perspective (e.g. by visualizing nuclei).

  • Especially for multi-color experiments, care should be taken when selecting counterstains to ensure that they are spectrally differentiable from the color of the antibody staining (e.g. DRAQ5 should be used as a nuclear counterstain rather than DAPI when using antibodies conjugated to blue emitting fluorochromes such as Alexa Fluor® 405 or DyLight® 405).
  • In the interest of time and for your convenience, we recommend using mounting media that contain counterstains.

The most commonly used chromogenic and fluorescent counterstains are shown below (adapted from (1)). 

Chromogenic counterstains
Color
Counterstain for
Hematoxylin (4 types - Harris’s, Mayer’s, Carazzi’s, and Gill’s)
Blue
Nucleus
Fast red
Red
Nucleus
Methylene blue
Blue
Nucleus
Methylene green
Blue/green
Nucleus
Toluidine blue
Blue
Nucleus

 

Fluorescent counterstains
Color
Counterstain for
Blue
Nucleus
DRAQ5™
Red
Nucleus
Hoechst 33258/33342
Blue
Nucleus
Phalloidin Variable; depending on which dye was conjugated to phalloidin
Filamentous
Actin
Propidium iodide
Blue/green
Nucleus
Sytox® Green
Green
Nucleus
Wheat germ agglutinin (WGA)
Variable; depending on which dye was conjugated to WGA
Plasma membrane

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Did we miss your favorite counterstain in the post? Then let us know.


References:

(1) Paul, C., Counterstaining for Immunohistochemistry: Choices, Choices
Available here (Accessed: December 02, 2015).


Alexa Fluor ® is a registered trademark of Molecular Probes Inc. OR, USA.
DyLight ® is a trademark of Thermo Fisher Scientific Inc. and its subsidiaries.
DRAQ5 ™ is a trademark of Biostatus Limited.
Sytox
® Green is a trademark of Life Technologies Company.

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