In order to obtain optimal fixation (and subsequent processing), please conform to the following recomendations
- Tissue size should not exceed 2.0 x 2.0 centimeters with an ideal thickness of three millimeters. Two to four millimeters is an acceptable range.
- Amount of fixative must be AT LEAST 10 times the volume of the sample; 15-20X is better.
- Fix for a MINIMUM of 24 hours; aim for 48 as a standard. A shaker or rocker will greatly assist in attaining even and complete fixation.
- Tissue can be submitted in formalin, but it is preferable that samples be transferred to 70% ethanol or PBS before submission. Please note the fluid type on the sample container.
- Use leak-proof flat bottom containers made for histology if possible. We can return these containers to you after processing to be washed and re-used. Avoid containers with narrow necks, 15mL falcon tubes for example, as tissue at the bottom of the tube may not have sufficient space to be adequately fixed.
Brain and other nervous tissue should be submitted in formalin after fixation for a minimum of 48 hours. Prolonged exposure to 70% ethanol (longer than 20 hours) can result in vacuole formation in the white matter, so transfer to ethanol is not recommended.
Eyes will exhibit superior morphology if fixed in Davidson’s Solution instead of formalin alone:
- Glacial acetic acid, 100 ml
- 95% ethyl alcohol, 300 ml
- 10% neutral buffered formalin, 200 ml
- Distilled water, 300 ml
After fixing in Davidson’s for 24 hours, eyes may be transferred to either formalin or 70% ethanol.
Tissues which are more prone to rapid autolysis should be collected first: pancreas, GI tract, liver and kidneys.
The intestinal tract should be gently flushed first with saline then formalin. Residual gut contents may hinder fixation and compromise the preservation of the mucosal architecture.
Liver and kidney should, at a minimum, be nicked to allow for better formalin penetration. Routine sampling of liver includes one cross-section of the median lobe and one from the left lateral lobe. For routine kidney trimming, bisect the left kidney longitudinally and the right kidney transversely.
Collect tissues immediately after euthanasia and place into fixative. This will prevent autolysis and drying. Dry edges or really any part of the tissue that dries out before being fixed can create artifacts demonstrated in stained sections.