Fix Your Samples
Amount of fixative: A general rule is a 1:10 sample:fixative volume.
Perfusion versus immersion: Tissue perfusion is ideal as the fixative is delivered via the vascular system and thus is rapid and complete. In some studies, perfusion may not be feasible (no access to fume hood, tissue samples are collected for other assays such as RNA). Please discuss your project with staff to determine what will be best for your project.
Time: Fixative duration is very important to ensure adequate fixation throughout the sample. For immersion fixation, allow ~1mm/hour for the fixative to fully penetrate tissues then additional hours to complete the fixation process. Consistency in fixation duration is important for reproducibility in immunolocalization assays between experimental groups. Overnight fixation is a common interval.
Temperature: Fixation in colder temperatures is slower so longer times may be needed.
Sample thickness: The goal is rapid fixation so all the sample fixes as quickly as possible. Specimens should be cut thin enough so that the fixative penetrates the tissue within a reasonably short time. The thickness and size of a nickel or 5mm is a good guide to use when trimming samples. When using immersion fixation, cut open samples to allow the fixative to penetrate the interior quickly as for the gastrointestinal track and kidney.
Overnight NBF fixation at room temperature or 24-48 hours in 4% PF under refrigeration are commonly used protocols for mouse tissues.
Trim
Great guidelines found here: RENI trimming guidelines. Trim samples and place in cassettes before submitting to the Core. Please contact core staff for assistance.
Cassette
Use only a #2 pencil for labeling cassettes or get printed cassettes. Solvents used in processing will remove ink labels made from most Sharpie markers. Keep your labeling legible and less than 12 characters.
To prevent small tissues from being lost during processing, place them in biopsy cassettes, with 1-2 sponges (avoid compression) or wrapped in filter paper. The paraffin processors use vacuum to facilitate tissue infiltration and this can coax small tissues out of regular cassettes.
Don’t overcrowd tissues in the cassette. Tissue that is compressed in the cassette will not adequately fix or infiltrate.
Submit your tissues in cassettes with the surface to be cut first placed down in the cassette or add comments to the submission form and tell histology staff about your specific orientation needs.
End fixation in your lab then transfer to PBS or 70% ethanol. Keep cassettes submerged.
Transport samples in a leak-proof container labeled with your name, PI and solution.
Frozen sample preparation
Samples can be snap-frozen in liquid nitrogen and placed directly into aluminum foil or they can be placed in cryomolds and embedded and frozen in OCT using dry ice and isopentane. Please wrap cryomolds in aluminum foil and label both cryomold and aluminum foil wrapper. Store at -80 until use. Transport on dry ice to avoid thawing.