Mouse Brain

Parvalbumin-tdTomato transgenic whole mouse brain

An Example Protocol from the Visikol® HISTO™ Protocol Builder

Materials

Reagent Volume
per specimen
Visikol® HISTO-1™ 20 mL
Visikol® HISTO-2™ 20 mL
Ethanol
Anhydrous (reagent grade)
110 mL
Phosphate buffered saline (PBS) 75 mL
Glass, polypropylene, or polyethylene containers with lids.
Polystyrene is not compatible with Visikol HISTO-2

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Important Imaging Considerations

  • Light sheet microscopes are ideal for imaging large volumes of tissue rapidly without causing photobleaching. However, Visikol HISTO reagents are not directly compatible with the water immersion objectives used in most light microscopes (e.g. Zeiss Z.1, OpenSPIM). See below for additional guidance on using an LSM with Visikol HISTO.
Note: Unless otherwise specified, all protocol steps should be performed at 4°C, in a closed container, with gentle shaking.


Parvalbumin-tdTomato transgenic whole mouse brain, captured with an Ultramicroscope II from LaVision Biotec.

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 50 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 50 mL PBS for at least 1 hour to ensure complete removal of the mounting media.

Tissue Clearing

  1. Treat tissue at 4°C with 50 mL 50% ethanol in PBS for 2 hours. Followed by 50 mL 70% ethanol in PBS for 2 hours.
  2. Treat tissue at 4°C with 50 mL 100% ethanol for 2 hours with gentle shaking.
  3. Remove from ethanol, make sure excess ethanol is absorbed with a Kimwipe™ or paper towel.
  4. Add at least 20 mL Visikol HISTO-1 to completely cover the sample. Incubate tissue at 4°C for 24 hours.
  5. Transfer to at least 20 mL Visikol HISTO-2 for 24 hours at 4°C 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.

Considerations for Light Sheet Microscopy

OpenSPIM Devices

For best results, samples should be imaged in Visikol HISTO-2. However, it will damage water dipping objectives present in many OpenSPIM setups. To avoid this, we suggest placing the specimen in a quartz, Simax, or glass cuvette filled with Visikol HISTO-2 then submerging the cuvette in the imaging chamber filled with 2,2’-thiodiethanol, which is compatible with water dipping objectives.

LaVision UltraMicroscope II

The UltraMicroscope is directly compatible with Visikol HISTO-2 and requires approximately 150 mL of additional reagent to completely fill the imaging chamber. Visikol HISTO-2 is hygroscopic and imaging quality will degrade in the open imaging chamber for long periods of time.

Zeiss Lightsheet Z.1

Visikol HISTO-2 is not directly compatible with the Zeiss Z.1, and requires special precautions. Please see our guide on using the Zeiss Z.1 with Visikol HISTO for more information.