Mammary Tissue

Mammary tumor from PyMT transfected mouse with Ki67 Cell Proliferation Labeling

An Example Protocol from the Visikol® HISTO™ Protocol Builder

Materials

Reagent Volume
per specimen
Visikol® HISTO-1™ 6 mL
Visikol® HISTO-2™ 6 mL
Penetration Buffer
(PBS / 0.2% Triton™ X-100 / 0.3 M glycine / 20% DMSO)
6 mL
10x Washing Buffer
(10x PBS with 2% Tween™ 20 and 100µg/mL heparin)
6 mL
Blocking Buffer
(PBS/0.2% Triton™ X-100 /6% donkey serum/10% DMSO)
3 mL
Antibody Buffer
(PBS/0.2% Tween™ 20/Heparin /3% donkey serum/5% DMSO)
6 mL
Primary and Secondary Antibodies
Nuclear stain
e.g. DAPI, Hoeschst, SYTOX, or TO-PRO stains
Methanol
Anhydrous (reagent grade)
80 mL
Phosphate buffered saline (PBS) 60 mL
PBS with 1% Triton™ X-100 12 mL
DMSO 2.4 mL
Glass, polypropylene, or polyethylene containers with lids.
Polystyrene is not compatible with Visikol HISTO-2

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Important! Obtaining a proper dilution factor for your antibody is the most critical variable in achieving good immunolabeling. For larger tissues, immunolabeling protocols should be optimized for dilution and incubation time prior to proceeding to larger tissues. Please follow our antibody validation protocol located in the guidebook.
Note: Unless otherwise specified, all protocol steps should be performed at room temperature, in a closed container, with gentle shaking.


Mammary tumor from PyMT transfected mouse with Ki67 Cell Proliferation Labeling. Captured with 10x air objective on an upright confocal microscope.

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 15 mL PBS solution for at least 1 hour before further procedures to remove traces of fixative.
  3. If tissues have been cryopreserved, wash an additional 3-5 times with 15 mL PBS for at least 1 hour to ensure complete removal of the mounting media.

Permeabilization

  1. Wash tissue at room temperature in 15 mL PBS for 45 minutes, then in 15 mL 50% methanol (in PBS) for 45 minutes, 15 mL 80% methanol in DI H2O for 45 minutes, and finally 15 mL 100% methanol for 45 minutes. Samples can be stored in methanol (preferably at 4ºC) indefinitely before proceeding with the next step.
  2. For highly pigmented tissues, results may be improved by bleaching samples by submerging in ice-cold 5% H2O2 in 20% DMSO/methanol (1 part 30% H2O2, 1 part DMSO, 4 parts methanol) and leaving at 4°C overnight. This step will significantly reduce impact of pigment in tissue.
  3. Wash samples at room temperature in 6 mL 20% DMSO/methanol for 45 minutes twice, then in 6 mL 80% methanol (in H2O) for 45 minutes, 6 mL 50% methanol (in PBS) for 45 minutes, 6 mL PBS for 45 minutes twice, and finally in 6 mL PBS/1% Triton™ X-100 for 45 minutes twice before further staining procedures.
  4. Incubate samples at room temperature in 6 mL Penetration Buffer for 2 hours with gentle shaking.
  5. Block samples in 3 mL Blocking Buffer at 37°C with gentle shaking for 13.5 hours.
  6. Incubate samples in a primary antibody dilution prepared in 3 mL Antibody Buffer and incubate at 37°C with gentle shaking for 13.5 hours.
  7. Depending on the specific antigen, an antibody dilution of 1:50 to 1:500 is typically required. Obtaining a proper dilution factor for your antibody is the most critical variable in achieving good labeling, please see our antibody validation protocol located in the guidebook for instructions on optimizing your antibody concentration. To prevent aggregates, we recommend centrifuging or passing the solution through a 0.45µm syringe filter prior to use.
  8. Be sure to dilute the 10x Washing Buffer to a 1X working concentration before using.
  9. Wash samples in 6 mL 1x Washing Buffer (5 times, 45 minutes each time, at 37°C, with gentle shaking).
  10. 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.
  11. Incubate in secondary antibody buffer (1:50 to 1:500 depending on dilution of primary antibody) prepared in 3 mL Antibody Buffer at 37°C with gentle shaking. Incubate samples for 13.5 hours.
  12. Wash in 6 mL 1x Washing Buffer (5 times, 45 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 15 mL 50% methanol in PBS for 45 minutes. Followed by 15 mL 70% methanol in PBS for 45 minutes.
  2. Treat tissue at room temperature with 15 mL 100% methanol for 45 minutes with gentle shaking.
  3. Remove from methanol, make sure excess methanol is absorbed with a Kimwipe™ or paper towel.
  4. Add at least 6 mL Visikol HISTO-1 to completely cover the sample. Incubate tissue at room temperature for 6 hours.
  5. Transfer to at least 6 mL Visikol HISTO-2 for 6 hours at room temperature to finish the clearing process.

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.