Paraffin Embedding
Tissues can be accepted in a variety of ways and costs are tailored to these methods.
Fresh tissue: If fresh tissue is submitted, it will be placed in 10% Neutral Buffered Formalin for fixation overnight prior to processing.
Fixed tissue: We accept tissue which has been fixed in a variety of fixatives. Once fixation is complete, please transfer samples to 70% Ethanol.
Fixed tissue in Cassettes: Customers are welcome to place their tissues in cassettes to ensure desired orientation.
The following are available to our customers for use and are available for pick-up in E1515:
- 10% Neutral Buffered Formalin (NBF)
- Cassettes (for a fee)
- Biopsy wraps/cassettes
To prepare cassettes:
- Fix tissue as desired: Allow ~1 mm/hour for the fixative to penetrate your tissues and a volume of 10-20 times fixative volume to tissue.
- Label cassettes: When labelling cassettes, use only a reagent resistive marker (ex: Statmark Pen) or a #2 hard lead pencil for cassettes, never a pen or Sharpie marker. Solvents used in processing can remove the ink from many "permanent" Sharpie markers.
- Ensure small pieces will not be lost: To prevent small tissues from being lost during processing, place in biopsy cassettes or wrap in filter paper. The tissue processor uses vacuum to facilitate infiltration which can remove tissue from the cassette.
- Place in cassettes in desired orientation: When tissue is placed into cassettes the tissue surface that is place down in the cassette will be placed down for embedding and this is the surface that is sectioned first. Please list any special embedding requests on submission form.
- Refrain from overcrowding. Tissue that is compressed in the cassette will not adequately fix, infiltrate, section, or stain.
- Specimens should be cut thin enough to allow adequate fixative penetration.
- Do not allow tissue to touch all sides of the cassette or become smashed in the lid.
- The thickness between 0.2 and 0.5 cm (approximately the size of a nickel) is a good guide to use when trimming samples.
- If batches of tissues are being submitted at the same time that vary significantly in size (whole organs vs biopsy samples), sort by size and submit in separate cassettes.
- Place specimens in spill roof container with a tight fitting lid. Be sure that all tissue and or cassettes are completely submerged in 70% Ethanol so samples do not dry out.
- Label the transport container: Please include the name of the PI, name of the Researcher (submitter), solution, and date.
- Provide samples and submission form to RHS. A separate sheet is also provided to list the specimen identification.
Tissue already in Paraffin Blocks or blank slides from paraffin blocks are gladly accepted for sectioning and staining.
Frozen Samples
Tissues can be accepted in a variety of ways and costs are tailored to these methods.
Rapid rather than slowly freezing reduces ice crystal formation in tissue.
Fresh tissue: If fresh tissue is submitted, rapidly frozen without fixation.
Fixed tissue: We accept tissue which has been fixed in a variety of fixatives. Some customers choose to prepare their samples through a sucrose gradient prior to freezing. Once tissue is prepared, please transfer samples to 30% sucrose prior to delivery to RHS.
Frozen blocks in OCT molds: Customers are welcome to freeze their own samples to ensure desired orientation. Frozen blocks can then be provided to RHS for sectioning/further processing.
To OCT embed your tissues:
- A pre-labeled tissue mold is filled one third full with OCT embedding compound.
- Tissue should be gently blotted free of extraneous fluid prior to being frozen.
- Tissue specimen is oriented in tissue mold (clearance between the edge of tissue and side of mold should be maintained)
- The remainder of tissue mold is filled with OCT embedding medium. Ensure that specimen is completely surrounded and covered by OCT.
- Tissue mold is floated on liquid nitrogen bath until completely frozen. OCT will be firm and opaque/white.
- Remove mold from liquid nitrogen and tightly wrap in foil.
- Place foil wrapped specimen in plastic bag that is properly labeled with specimen identification and date. Please include the name of the PI, name of the Researcher (submitter), solution, and date.
- Tissue should be stored at -80 C and delivered to RHS on dry ice.
- Provide samples and submission form to RHS. A separate sheet is also provided to list the specimen identification.