As tissue samples are collected, ideally the time to fixation should be within a 20-minute window. The quicker the better, because stress responses are initiated almost immediately after the tissue is deprived of its blood supply. If you need to, keeping tissue near 4°C prior to fixation will slow down some of these processes induced by oxygen and nutrient deprivation.

Prior to fixation, tissue should be maintained in a neutral isotonic solution with physiologic calcium and magnesium concentrations to avoid any unnecessary disruptions of adhesion systems and activation of signaling cascades. An example of this is PBS (phosphate-buffered saline) with calcium and magnesium.

Besides time to fixation, the following factors in the pre-analytics stage can all impact tissue quality:

Temperature of fixative
Type of fixative
Volume of fixative
Total time in fixative
Size of tissue sample

There are several types of fixatives on the market, including PFA (paraformaldehyde) and NBF (neutral buffered formalin).

For the volume of fixation needed, the general rule is 10-20x the size of your tissue. For many research studies a 4 oz., 8 oz., or 16 oz. container will be sufficient. When you are working with large organs you may need larger containers that so that the fixative (and subsequent reagents) can access both the exterior and interior of the organ.

Formalin is designed to penetrate rapidly and fix slowly. You’ll want to make sure to allow for adequate timing:

Small biopsies: 12-18 hours fixation time
Standard tissue sections (1.5 x 1.5 x 0.4 cm): 24-72 hours fixation time

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