Optimizing staining protocols with Visikol HISTO clearing: DAPI vs Hoescht

In image-based cellular analysis, nuclear counterstains can offer crucial information by providing a baseline total cell count for quantitatively assessing secondary label positivity or for determining the subcellular localization of such secondary labels. Therefore, it is important to utilize an appropriately vetted nuclear counterstain, particularly when chemically clearing 2D or 3D cell cultures. Even more generally, careful optimization of any labeling and clearing procedures yields the best results when translating to an experimental setting.

  DAPI and Hoescht labeling of HepG2 cells cultured in 2D. Exposure times indicated in white italic text. Cleared versus uncleared images were identically contrast adjusted for comparison, except where indicated otherwise by asterisk (to enable visualization of dimmer cell fluorescence).

DAPI and Hoescht labeling of HepG2 cells cultured in 2D. Exposure times indicated in white italic text. Cleared versus uncleared images were identically contrast adjusted for comparison, except where indicated otherwise by asterisk (to enable visualization of dimmer cell fluorescence).

Throughout the optimization process 2D cell culture offers the ability to verify label specificity and compatibility with various reagents in your protocol, independent of the complexities that might be introduced by 3D culture. For example, necessary adjustments to laser power or exposure time with clearing in 3D models can make it challenging to distinguish issues with your staining protocol from those associated with imaging optimization. Yet, in 2D models, since the cell layer is relatively thin compared to 3D culture, clearing should have a minimal effect on the necessary laser powers or exposure times required to visualize your label. 2D models also eliminate the need to optimize the labeling procedure to account for 3D model penetration, thus offering an all-around simpler approach to initially screening and optimizing your labeling procedure prior to translating into higher order models.

We have implemented such 2D cell culture models to provide a validated recommendation for a nuclear counterstain. Based on feedback from our customers, we compared the use of both DAPI and Hoescht to stain nuclei and found DAPI to outperform Hoescht when clearing is required. Since staining is typically performed in aqueous mediums and Visikol® HISTO-M™ is a non-aqueous solution, clearing can yield suboptimal labeling with certain small molecule dies. We found this to be the case for Hoescht, but DAPI was unaffected by clearing. Even at dilutions as low as 1:5000, cleared 2D cell cultures exhibited higher contrast in fluorescence intensity versus background when compared to uncleared tissues. We have found that these recommendations hold true in a variety of 3D cell cultures as well. For these reasons, we recommend choosing DAPI as a nuclear counterstain when clearing tissues for enhanced image quality.

   DAPI labeled spheroids   . Top row: Uncleared (left) versus cleared (right) HepG2 3D cultures, labeled with DAPI (blue), Thermo fixable Live/Dead stain (green) and Ki67 (red). Bottom row:  Stemonix microBrain 3D  (left) and  Cellaria Wood Breast Cell model , grown in 3D culture (right), labeled with DAPI. All images are z-projections, all scale bars 50   µ  m.

DAPI labeled spheroids. Top row: Uncleared (left) versus cleared (right) HepG2 3D cultures, labeled with DAPI (blue), Thermo fixable Live/Dead stain (green) and Ki67 (red). Bottom row: Stemonix microBrain 3D (left) and Cellaria Wood Breast Cell model, grown in 3D culture (right), labeled with DAPI. All images are z-projections, all scale bars 50 µm.

Michael Johnson