3D microscopy tissue section

Improve 3D cell imaging results

The Invitrogen CytoVista Clearing Reagents are recommended when imaging thick tissues (up to 10 mm) and 3D cell cultures of various thickness, including organoids and spheroids. The CytoVista reagents are optimized for clearing and mounting these challenging samples in order to enable optimal clarity for sharp, bright fluorescent images. The reagents are provided in kitted and standalone formats to meet your experimental needs.

Why standard mountants won’t work for imaging 3D cell culture and thick tissue

The imaging of organoids, spheroids and thick tissue presents new challenges inherent in the samples themselves. Because of the opaqueness of tissue and the opaqueness of 3D cell cultures, it is harder to obtain sharp images with great clarity using standard imaging reagents and techniques. A fluorescently labeled thick specimen must be “cleared” so that the sample is more transparent for easier 3D imaging. The Invitrogen CytoVista Clearing Reagents are optimized for tissue up to 10 mm thick, and spheroids and organoids of varying thickness.

Enable sharp and bright 3D fluorescent images with CytoVista reagents

When staining adherent cells, all that is required is the addition of a simple staining buffer, but a fluorescently labeled thick specimen must be optically “cleared” to make it more transparent for easier three-dimensional imaging. Ideally clearing reagents should have minimum effect on a sample’s morphology or reagent fluorescent sensitivity. The CytoVista reagents for 3D imaging provide both optical clearing and mounting functions to meet these needs.

These CytoVista clearing reagents provide:

  • Rapid clearing—1 mm thick samples can be cleared in as little as 2 hours and whole mouse brains of 8 mm thickness can be cleared in as little as 24 hours
  • Gentle optical clearing—Minimal effect of detection sensitivity or sample morphology is observed after fluorescence labeled–tissue is cleared for 3D imaging
  • Workflow compatibility—Easily adapt use of the reagents to IHC workflows without additional equipment or instruments
  • Platform compatibility—Works with most fluorophores and standard fluorescence imaging instruments
  • Flexibility—Store optically cleared tissue samples and cultures for weeks with minimal effect on sample morphology or fluorophore brightness and specificity
  • Reversibility—For precious samples, the clearing can be reversed and the tissue can be processed and sectioned for histology studies such as hematoxylin and eosin (H&E) staining

CytoVista reagent selection guide

 CytoVista 3D Cell Culture Clearing / Staining KitCytoVista Tissue Clearing / Staining Kit
UseReady-to-use, fixed-cell soft-setting imaging reagent for immediate imaging
Sample typeOrganoids and spheroids up to 1 mm thickTissue samples up to 10 mm thick
Refractive index1.481.53
Kit contents
  • 3D cell culture clearing reagent
  • Antibody dilution buffer
  • Blocking buffer
  • Antibody penetration buffer
  • 10X wash buffer
  • Tissue clearing reagent
  • Tissue clearing enhancer
  • Antibody dilution buffer
  • Blocking Buffer
  • Antibody penetration buffer
  • 10X wash buffer
  • Tissue permeabilization buffer
Cat. No.V11325V11324

*Shipped and stored at multiple temperatures. Store according to product labeling

CytoVista Clearing Reagents

ReagentSample typeRefractive indexCat. No.Size
3D Cell Culture Clearing ReagentOrganoids Spheroids up to 1 mm thick1.48V1132610mL
V1131530 mL
V11316100 mL
Tissue Clearing ReagentTissue samples up to 1 mm thick1.50V1130030 mL
V11301100 mL
Tissue Clearing EnhancerTissue samples up to 10 mm thick1.53V1130230 mL
V11303100 mL
Tissue Clearing Reagent & EnhancerTissue samples up to 10 mm thick1.53V1130410 mL of each
V1132230 mL of each
V11323100 mL of each

CytoVista Buffers

BufferCat. No.Size
Antibody Dilution BufferV1130530 mL
V11306100 mL
Blocking BufferV1130730 mL
V11308100 mL
Antibody Penetration BufferV1130930 mL
V11310100 mL
Wash BufferV1131170 mL
V11312200 mL
Tissue Permeabilization BufferV1131330 mL
V11314100 mL

CytoVista Tissue Imaging Chambers

DepthCat No.Size
0.75 mm deepV11333Set of 6
1.75 mm deepV11317Set of 6
3.5 mm deepV11318Set of 6
7 mm deepV11319Set of 6

CytoVista Mouse Brain Slicers

TypeCat No.Size
1 mm Coronal SlicesV113351 each
1 mm Sagittal SlicesV113341 each
2 mm Coronal SlicesV113201 each
2 mm Sagittal SlicesV113211 each

Image gallery of 3D cell culture and tissue clearing applications

Imaging spheroids

Comparison of a spheroid imaged in 10 slices. Non-cleared tissue core image is not seen in the sphereoid, whereas it is visible easily in the cleared spheroid

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.

Figure 2. Obtain clearer images of spheroids with CytoVista 3D Cell Culture Clearing Reagent. The CytoVista 3D Cell Culture Clearing Reagent cleared HepG2 spheroids to minimize light scattering, resulting in a sharper image and a more comprehensive cell count. By accurately counting dead cells at the core of the spheroid, more viable cells can be analyzed. Confocal z-slices of (A) uncleared and (B) cleared HepG2 spheroids. Blue = DAPI; Green = LIVE/DEAD Fixable Green Dead Cell Stain.

Cell proliferation within a spheroid

Figure 3. Analyze more cells for proliferation markers when the spheroid is cleared CytoVista 3D Cell Culture Clearing Reagent. Ki-67 and Click-iT EdU Cell Proliferation Kit for Imaging Alexa Fluor 488 dye were used as markers for proliferation. More cells were analyzed in the cleared samples as compared to the uncleared sample due to the effect of light scattering from uncleared samples. HeLa spheroids were labeled with Hoechst 34580 (in blue), Click-iT EdU Cell Proliferation Kit for Imaging Alexa Fluor 488 dye (in green), and Ki-67 MAb, Alexa Fluor 647 conjugate. Cells were imaged using a Thermo Scientific high-content confocal imaging system.

High-content screening video of a Stemonix 3D MicroBrain model

Video 1. Prepare detailed, 3D composite videos using CytoVista 3D Cell Culture Clearing/Staining Kit. A Stemonix 3D MicroBrain model, derived from iPSC-derived neural progenitor cells, was imaged used a Thermo Scientific CellInsight CX7 LZR High-Content Analysis Platform. The tissue was cleared, mounted and imaged in CytoVista 3D Cell Culture Clearing/Staining Kit. Invitrogen NeuroTrace Green Fluorescent Nissl Stain was used with a GFAP primary antibody, and a Goat Anti-Rabbit IgG Alexa Fluor Plus 647 secondary antibody. ImageJ was used to generate the .avi files.

3D, high-content screening video of human lung tissue

Video 2. See clear & sharp 3D fluorescent videos images with CytoVista Tissue Clearing/Staining Kit. A 7.0 mm x 5.0 mm X 0.612 mm piece of human lung tissue was labeled with α-SMA, CD31, and DAPI (DAPI=Blue, SMA=Red, CD31=Green) nuclear stain with primary + secondary antibody steps. After labeling, the tissue was cleared and mounted using CytoVista Tissue Clearing/Staining Kit, and imaged with the Thermo Scientific CellInsight CX7 LZR High-Content Analysis Platform. The video shows a sweep through one of fields taken through this tissue using a 10X objective on a CellInsight CX7 LZR confocal system. The human lung tissue was imaged using a 1.53 um Z-step size to achieve cubic voxels.

3D confocal microscopic image of tissue

Video 3. Get beautiful three-dimensional view of tissue with CytoVista Tissue Clearing/Staining Kit. Three-dimensional high content confocal microscopic image showing the anterior view of murine prostate lobe cleared and mounted with CytoVista Tissue Clearing/Staining Kit. Tissue was labeled with E-cadherin (red), Beta-3 tubulin (green) CD31 (white) along with a murine urethra labeled with E-cadherin (red) and CD31 (white) to evaluate.

3D imaging of 1 mm thick tissue

Figure 4. Easily image thick tissue samples using the CytoVista 3D Cell Culture Clearing Reagent. (A) A rat brain sample, 1 mm thick, was labeled with an anti-parvalbumin antibody. Captured with 10x air objective on an upright confocal microscope. (B) A mammary tumor sample, 1 mm thick obtained from a PyMT-transfected mouse, was labeled with an anti-Ki-67 FITC conjugated monoclonal antibody to study cell proliferation labeling. Captured with 10x air objective on an upright confocal microscope.

Whole mouse brain imaging

Figure 5. Obtain clear, sharp images from whole mouse brain samples using the CytoVista Tissue Clearing / Staining Kit.
(A) Parvalbumin-tdTomato transgenic whole mouse brain, captured with light sheet microscope. (B) Whole mouse brain cleared with CytoVista Tissue Clearing / Staining Kit and imaged with light sheet microscope.

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