- Fix tissue with 4% PFA overnight at 4oC followed by one hour at room temperature.
- Wash tissue in at least 10 mL PBS solution for at least 1 hour before further procedures to remove traces of fixative.
- After fixation and washing, remove and transfer to PBS with 0.05% sodium azide for indefinite storage.
NOTE: If tissues have been cryopreserved, wash an additional 3-5 times with 10 mL PBS for at least 1 hour to ensure complete removal of the mounting media.
NOTE: For tissues that contain fluorescent protein, replace methanol with ethanol and conduct steps involving ethanol at 4oC.
- 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.
NOTE: 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.
- 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.
- Incubate samples at room temperature in 6 mL Visikol HISTO Antibody Penetration Buffer for 2 hours with gentle shaking.
- Block samples in 3 mL Visikol HISTO Blocking Buffer at 37°C with gentle shaking for 13.5 hours.
- Incubate samples in a primary antibody dilution prepared in 2 mL Visikol HISTO Antibody Dilution Buffer and incubate at 37°C with gentle shaking for 13.5 hours. For labeling with anti-Ki67, use a 1:100 dilution (Thermo, Cat # RB-1510).
NOTE: 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. We suggest that labeling is optimized starting in thin tissues using the below approach before proceeding to thicker tissues.
1. Cut three to five 100-200 µm tissue sections to explore dilutions ranging from 1:500 – 1:50. Smaller sections can be stained and cleared in a single workday
2. Cut perpendicular cross-sections of the tissue slice to examine evenness and depth of penetration of stain.
3. Antibody concentration is a balancing act: too low and there will be low signal to noise, too high and the outer layers will “shadow” the inner layers due to absorbance in the outer layers.
4. Volume of antibody solution should completely cover tissue of interest
To prevent aggregates, we recommend centrifuging or passing the solution through a 0.45µm syringe filter prior to use.
- Be sure to dilute the Visikol HISTO 10X Wash Buffer to a 1X working concentration before using.
- Wash samples in 6 mL Visikol HISTO 1X Wash Buffer (5 times, 45 minutes each time, at 37°C, with gentle shaking).
- 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. We recommend using DAPI at a dilution of 1:1000 for a nuclear counterstain on 1 mm tissue sections.
- Incubate in secondary antibody buffer prepared in 3 mL Visikol HISTO Antibody Dilution Buffer at 37°C with gentle shaking for 13.5 hours. The secondary antibody (Goat anti-Rabbit IgG Alexa Fluor Plus 488, Thermo Cat # A32731) should be used at a dilution of 1:200. We recommend filtering the secondary antibody labeling solution with a syringe filter to minimize background staining and non-specific fluorescence.
- Wash in 6 mL Visikol HISTO 1X Wash 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.
- Treat tissue at room temperature with 15 mL 50% methanol in PBS for 45 minutes. Followed by 10 mL 80% methanol in DI H2O for 45 minutes.
- Treat tissue at room temperature with 16 mL 100% methanol for 45 minutes with gentle shaking.
- Remove from methanol, make sure excess methanol is absorbed with a Kimwipe™ or paper towel.
- Add at least 6 mL of Visikol HISTO-1 to completely cover the sample. Incubate tissue at room temperature for 6 hours.
- Transfer to at least 6 mL of Visikol HISTO-2 for 6 hours at room temperature to finish the clearing process.
NOTE: Do not treat specimens with other media after clearing. For optimal results, imaging should be performed directly in Visikol HISTO-2. The Visikol HISTO-2 solution contains anti-fade agents, and specimens cleared with Visikol HISTO-2 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.
● Place tissue into ClearWell of an appropriate thickness or another similar imaging cuvette and fill with Visikol HISTO-2.
● After 3D imaging, if desired, additional histological or biochemical assays can be performed on Visikol HISTO treated tissue by reversing the clearing. This can be simply done by washing the tissue three times with 10 mL 100% ethanol at room temperature. The tissue can then be processed as any fixed tissue, for SDS-PAGE, Western Blotting, or embedding, sectioning, and staining with H&E, Nissl, IHC or other histological stains.