Tips for Step 18 – Counterstain

Fig. 3. PureBlu™ Nuclear Fluorescent Dye Hoechst 33342.

Fig. 3. PureBlu™ Nuclear Fluorescent Dye Hoechst 33342. Paraffin section of human colon adenocarcinoma (heat induced antigen retrieval with citrate buffer
(pH 6), blocked with 10% FCS) stained with Mouse Anti-Human Cytokeratin 18 Antibody (MCA1864H, green). Nuclei were counterstained with PureBlu Dye Hoechst 33342 (1351304, blue).

Fig. 4. PureBlu Dye DAPI.

Fig. 4. PureBlu Dye DAPI. Paraffin section of human pancreas stained with Guinea Pig Anti-Pig Insulin Antibody (5330-0104G, red), Mouse Anti-Human Chromogranin A Antibody (MCA4773, green) and PureBlu Dye DAPI (1351303, blue).

Counterstains provide contrast as well as knowledge about localization within the tissue sample (for instance by visualizing nuclei or filamentous actin).

What to consider when selecting counterstains?

  • Ensure that the counterstain can be easily spectrally distinguished from your precipitate color or fluorescent label. For example, when using red emitting fluorophores, select a blue nuclear counterstain such as DAPI or Hoechst 33342 (Tables 4 and 5)

  • To save time, consider using pre-made mounting media already containing counterstains

Table 4:  Commonly used chromogenic counterstains.

Chromogenic Counterstains


Counterstain for



Proteins containing cationic groups (Kim et al. 2016)

Fast Red/Kernechtrot



Hematoxylin (4 types - Harris’s, Mayer’s, Carazzi’s, and Gill’s)



Methylene blue



Methylene green



Toluidine blue



Adapted from Kim et al. 2016 and Paul no date. Fast red/Kernechtrot has been included in the table according to IHCWorld (no date c).

Table 5:  Commonly used fluorescent counterstains.

Fluorescent Counterstains Colors Counterstain for
DAPI Blue Nuclei
DRAQ5 Red Nuclei
Hoechst 33258/33342 Blue Nuclei
Propidium iodide Red Nuclei
SYTOX Green Green Nuclei
Phalloidin Conjugate dependent Filamentous actin