Clearing Reagents

Improve 3D cell imaging results

The Invitrogen CytoVista 3D Culture Clearing Reagents are optimized for 3D tissues, spheroids, organoids, and cell cultures of various thickness. We have various solutions to suit your experimental needs. Tissues sections 1 mm thick can be cleared for imaging in as little as 2 hours, and whole mouse brains of 8 mm thickness can be cleared in as little as 24 hours.

CytoVista product highlights

  • Rapid clearing of fluorescence labeled–tissue for 3D imaging with minimal effect on detection sensitivity or sample morphology (Figure 1)
  • Can be easily adapted to IHC workflows without additional equipment or instruments
  • Cleared tissue can be reversed and processed for histology studies, including haemotoxylin and eosin (H&E) staining
  • Compatible with most fluorophores, including dyes and fluorescent proteins
  • Compatible with standard fluorescence imaging instruments such as wide-field microscopes, high-content analyzers, confocal microscopes, and light sheet microscopes
  • May be used to store and stabilize 3D tissue and cultures for weeks with minimal effect on sample morphology or fluorophore brightness and specificity

Comparison of mounting media, antifade reagents, and 3D clearing reagents

  ProLong Glass ProLong Diamond ProLong Gold ProLong Live SlowFade Diamond SlowFade Gold CytoVista 3D Cell Culture Clearing / Staining Kit CytoVista Tissue Clearing / Staining Kit
Used without CytoVista Tissue Clearing Enhancer Used with
CytoVista Tissue Clearing Enhancer)
  Learn more Learn more Learn more Ordering information Ordering information
General specifications
Form Ready-to-use hard-setting mountant 100x concen-trated liquid Ready-to-use soft-setting mountant Ready-to-use soft-setting imaging reagent
Refractive index 1.52 (after curing) 1.47 (after curing) 1.47 (after curing) 1.3 1.42 1.42 1.48 1.50 1.53
What type of imaging are you planning?
Live-cell imaging                  
Fixed-cell, immediate imaging                  
Fixed-cell, long-term imaging (up to 10 µm sample thickness)                  
Fixed-cell, long-term imaging (up to 150 µm sample thickness)                  
Organoids/ spheroids                  
Up to 5 mm thickness                  
Up to 10 mm thickness                  
Which type of microscope objective is most recommended? (All objective types are compatible with these reagents)
Oil immersion objective                  
Glycerol-corrected objective                  
Water- or air-corrected objective                  
What fluorophores does your sample include?
Alexa Fluor dyes                  
Traditional dyes                  
Fluorescent proteins                  
Do you want to add counterstain in the mountant?
Counterstain with DAPI                  
No counterstain                  
CytoVista 3D Cell Culture Clearing/Staining Kit

Figure 1. CytoVista 3D Cell Culture Clearing/Staining Kit reduces light scattering and improves the number of cells that can be counted, especially at the core of the spheroid.  Montage of slices from confocal image z-stack, nuclei stained with SYTOX Green dye; (A) Non-cleared microtissue; (B) Cleared microtissue; Z-projections are outlined in white; z-projections mask difficulty of imaging non-cleared microtissues since z-projections hide the eclipsing that occurs in imaging. Cleared microtissues show no eclipsing, and interior cells are visible.


Applications for 3D culture clearing and Imaging

Application example 1: Viability (live/dead determination)

As demonstrated with HepG2 spheroids in Figure 2, CytoVista 3D Culture Clearing Reagent cleared the spheroid to minimize light scattering, resulting in clearer images and a more comprehensive cell count. By accurately counting dead cells at the core of the spheroid, more viable cells can be analyzed.

CytoVista 3D Cell Culture Clearing/Staining Kit

Figure 2. CytoVista 3D Cell Culture Clearing/Staining Kit helps deliver clearer images and a more comprehensive cell count.  Confocal z-slices of (A) cleared and (B) uncleared HepG2 spheroids. (C) Quantification of dead cell frequencies and percentages of dead (DAPI labeled) cells.  Blue = DAPI; Green = LIVE/DEAD Fixable Green Dead Cell Stain. Scale bar 100 µm. Mean values ± SEM for independently imaged spheroids shown in data;

* indicates p<0.05 for two-tailed two-sample t-test between cleared and uncleared means.

Application example 2: Cell proliferation within the spheroid

As shown in Figure 3, Ki67 and Click-it EdU were used as markers for proliferation. Due to the effect of light scattering from uncleared samples, we’ve demonstrated that more cells can be analyzed when the spheroid is cleared with CytoVista 3D Culture Clearing Reagent.

HeLa spheroid

Image gallery

CytoVista-img-3a
1 mm thick rat brain sample labeled with an anti-parvalbumin antibody. Captured with 10x air objective on an upright confocal microscope.
CytoVista-img-3b
1 mm thick mammary tumor sample from a PyMT-transfected mouse with anti-Ki-67 antibody cell proliferation labeling. Captured with 10x air objective on an upright confocal microscope.
CytoVista-img-3c
Parvalbumin-tdTomato transgenic whole mouse brain, captured with light sheet microscope.
CytoVista-img-3d

Whole mouse brain cleared with CytoVista 3D Cell Culture Clearing/Staining Kit and imaged with light sheet microscope.


CytoVista kits

  CytoVista 3D Cell Culture Clearing / Staining Kit CytoVista Tissue Clearing / Staining Kit
  Cat. No. V11325 Cat. No. V11324
CytoVista Tissue Clearing Reagent   30 mL
CytoVista Tissue Clearing Enhancer   30 mL
CytoVista 3D Cell Culture Clearing Reagent 30 mL  
CytoVista Antibody Dilution Buffer 30 mL 30 mL
CytoVista Blocking Buffer 30 mL 30 mL
CytoVista Antibody Penetration Buffer 30 mL 30 mL
CytoVista 10X Wash Buffer 70 mL 70 mL
CytoVista Tissue Permeabilization Buffer   30 mL
Storage Shipped and stored at multiple temperatures. Store according to product labeling.

CytoVista reagents and accessories (a la carte)

Product Cat. No. Size Notes Storage
CytoVista Tissue Clearing Reagent V11300 30 mL Clearing and imaging reagent, with refractive index of 1.50
  • Store at room temperature in dry environment. 
  • Do not Freeze. 
  • When stored as directed, the product is stable for 6 months after receipt.
V11301 100 mL
CytoVista Tissue Clearing Enhancer V11302 30 mL Clearing and imaging reagent, with refractive index of 1.53
V11303 100 mL
CytoVista Tissue Clearing Reagent + Enhancer V11304 10 mL Reagent 10 mL Enhancer Clearing and imaging reagent, with refractive index of 1.53
V11322 30 mL Reagent 30 mL Enhancer
V11323 100 mL Reagent 100 mL Enhancer
CytoVista 3D Cell Culture Clearing Reagent V11315 30 mL Clearing and imaging reagent, with refractive index of 1.50
V11316 100 mL
CytoVista Antibody Dilution Buffer V11305 30 mL PBS/0.2% Tween  20/Heparin/3% donkey serum/5% DMSO
  • Store at 2-8°C. 
  • When stored as directed, the product is stable for 6 months after receipt.
V11306 100 mL
CytoVista Blocking Buffer V11307 30 mL PBS/0.2% Triton™ X-100 /6% donkey serum/10% DMSO.
V11308 100 mL
CytoVista Antibody Penetration Buffer V11309 30 mL PBS / 0.2% Triton™  X-100 / 0.3 M glycine / 20% DMSO
V11310 100 mL
CytoVista 10X Wash Buffer V11311 70 mL PBS with 0.2% Tween-20 and 10µg/mL heparin. 
V11312 200 mL
CytoVista Tissue Permeabilization Buffer V11313 30 mL Not Provided
V11314 100 mL
CytoVista Tissue Imaging Chamber (1.75 mm Deep) V11317 Set of 6 NA
  • Store at room temperature Do not Freeze. 
  • When stored as directed, the product is stable for 6 months after receipt.
CytoVista Tissue Imaging Chamber (3.5 mm Deep) V11318 Set of 6 NA
CytoVista Tissue Imaging Chamber (7 mm Deep) V11319 Set of 6 NA
CytoVista 2 mm Coronal Mouse Brain Slicer V11320 1 each NA
CytoVista 2 mm Sagittal Mouse Brain Slicer V11321 1 each NA

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