Organoid

NCI2170 Lung Cancer Organoid with Ki67 Cell Proliferation Labeling

An Example Protocol from the Visikol® HISTO™ Protocol Builder

Materials

Reagent Volume
per specimen
Visikol® HISTO-M™ 200 μL
Penetration Buffer
(PBS / 0.2% Triton™ X-100 / 0.3 M glycine / 20% DMSO)
200 μL
10x Washing Buffer
(10x PBS with 2% Tween™ 20 and 100µg/mL heparin)
200 μL
Blocking Buffer
(PBS/0.2% Triton™ X-100 /6% donkey serum/10% DMSO)
200 μL
Antibody Buffer
(PBS/0.2% Tween™ 20/Heparin /3% donkey serum/5% DMSO)
400 μL
Primary and Secondary Antibodies
Nuclear stain
e.g. DAPI, Hoeschst, SYTOX, or TO-PRO stains
Methanol
Anhydrous (reagent grade)
953 μL
Phosphate buffered saline (PBS) 1 mL
PBS with 1% Triton™ X-100 400 μL
DMSO 113 μL
Rounded, clear bottom well plate with lid.
We recommend Corning® Cat. 4515.

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Important Imaging Considerations

  • Significant loss of signal from optical attenuation limits the depth of imaging attainable with air objectives to 0.5-1mm. We recommend using smaller tissue portions for air objectives, or using software compensation to increase laser power as depth into tissue increases.
  • With air objectives and an inverted microscope, imaging depth will be limited to approximately 500 μm. Imaging depth can be increased by increasing laser power with increasing depth into tissue
Note: For 3D cell cultures, all volumes in this protocol will assume a standard 96-well plate with a 200 μL working volume. Adjust volumes accordingly if you are using another plate style.
Note: Unless otherwise specified, all protocol steps should be performed at room temperature, in a closed container, with gentle shaking.


NCI 2170 lung cancer organoid, Nuclei labeled with SYTOX (blue), proliferating cells labeled with anti-Ki67 (red). Captured with Thermo Scientific CX7 HCS System.

Tissue Processing

  1. Fix tissue in your chosen fixative. After fixing for 24 hours, remove and transfer to PBS with 0.05% sodium azide for indefinite storage.
  2. Wash tissue in at least 200 μL PBS solution for at least 15 minutes before further procedures to remove traces of fixative.
  3. If tissues have been cryopreserved, wash an additional 3-5 times with 200 μL PBS for at least 15 minutes to ensure complete removal of the mounting media.

Permeabilization

  1. Wash tissue at room temperature in 200 μL PBS for 2 minutes, and finally 200 μL 100% methanol for 2 minutes. Samples can be stored in methanol (preferably at 4ºC) indefinitely before proceeding with the next step.
  2. Wash samples at room temperature with 200 μL 20% DMSO/methanol for 2 minutes.
  3. Wash samples at room temperature with 200 μL methanol for 2 minutes.
  4. Wash samples at room temperature with 200 μL PBS/1% Triton™ X-100 for 2 minutes
  5. Incubate samples at room temperature in 200 μL Penetration Buffer for 15 minutes with gentle shaking.
  6. Block samples in 200 μL Blocking Buffer at 37°C with gentle shaking for 15 minutes.
  7. Incubate samples in a primary antibody dilution prepared in 200 μL Antibody Buffer and incubate at 37°C with gentle shaking for 30 minutes.
  8. Depending on the specific antigen, an antibody dilution of 1:50 to 1:500 is typically required. To prevent aggregates, we recommend centrifuging or passing the solution through a 0.45µm syringe filter prior to use.
  9. Be sure to dilute the 10x Washing Buffer to a 1X working concentration before using.
  10. Wash samples in 200 μL 1x Washing Buffer (5 times, 5 minutes each time, at 37°C, with gentle shaking).
  11. Add desired nuclear label to the secondary antibody buffer at a dilution of 1:100 – 1:5000 depending on the results of your optimization experiments.
  12. Incubate in secondary antibody buffer (1:50 to 1:500 depending on dilution of primary antibody) prepared in 200 μL Antibody Buffer at 37°C with gentle shaking. Incubate samples for 30 minutes.
  13. Wash in 200 μL 1x Washing Buffer (5 times, 5 minutes each time, at 37°C, with gentle shaking). Samples may be kept in this solution indefinitely before proceeding with further steps.

Tissue Clearing

  1. Treat tissue at room temperature with 200 μL 100% methanol for 2 minutes with gentle shaking.
  2. Remove methanol from the well.
  3. Add 200 μL Visikol HISTO-M to the well and proceed directly to imaging.

Imaging Guidance

Warning! Do not treat specimens with other media after clearing. For optimal results, imaging should be performed directly in Visikol HISTO reagents. Visikol HISTO solutions contain anti-fade agents, and specimens cleared with Visikol HISTO do not need to be mounted in an additional anti-fade media. Other solutions (particularly aqueous media) will cloud the tissue or completely reverse the clearing, interfering with 3D volume imaging. Once the cleared tissues have been successfully imaged, see below for additional information on reversing the clearing to perform downstream assays.