Tips about Fixation and Formalin

Contact: Julie Randolph-Habecker
Contact phone: (206) 667-6845

Training Type: Self-Directed
Training Format: How-to

The function of fixation is to inhibit the decay or autolysis of tissue so that samples may be preserved for future study. The quality of the fixation can greatly impact the usefulness of that tissue. Because tissue samples are valuable, several factors should be considered during the planning stages of an experiment.  (Adapted from Freida Carson Histology: A Self-Instructional Text, second edition, 1997)

The Experimental Histopathology Shared Resources cannot guarantee the quality of specimens that have been improperly fixed.

Temperature:

Room Temperature

Generally, fixation at room temperature is sufficient to maintain excellent morphological detail. An increase in temperature can increase the rate of fixation but can also increase the rate of autolysis. However, it has been reported that fixation temperature of 45OC has little effect on tissue morphology.

Size:

No thicker than a nickel (3 – 5 mm) and no larger than a postage stamp (2cm)

Fixative penetration is usually directly related to tissue thickness. If specimens are very large, it takes a great deal of time for the fixative to penetrate the inner portion of the sample. As a result, autolysis can occur in the center of thick tissues. This can be amplified if the tissue is covered by a relatively impermeable membrane. As a result, the tissue must remain in fixative for an extended period of time, which can cause uneven fixation and affect techniques such as immunohistochemistry. If the sample is smaller or opened, allowing the fixative greater access to the tissue, these problems can be avoided.

As a guideline, consider that formalin penetration is slow, approximately 0.5mm/hr. Most references recommend tissue should be no larger than 3 – 5 mm.

Volume Ratio:

The fixative volume should be at least 15 to 20 times greater than the tissue volume

The most common error we see in our laboratory is insufficient ratio of tissue volume to fixative volume. We recommend that the fixative volume should be at least 15 to 20 times greater than the tissue volume. As the fixative molecules bind to the tissue, they are eventually depleted. This often causes poor fixation and can result in staining artifacts.

Please use an appropriate container when delivering samples to the facility. For safety reasons, the shared resources staff will no longer remove samples that have been forced into containers and samples will be returned unprocessed.

Time:

Place specimens in fixative immediately after removal; fix a 5mm piece of tissue for a minimum of 72 hours.

In order to maintain tissue morphology, samples should be fixed immediately after removal or death. The longer the blood supply is interrupted, the poorer the quality of tissue.

Fixation is the single most important preparative histological technique. Poor fixation cannot be remedied at any later stage and is the source of most of the problems we see in our lab. In our experience, under-fixation is a much greater problem than over-fixation.

Adequate fixation time is critical for accurate morphology. Under-fixed tissue can produce artifacts from subsequent dehydrating alcohols used in processing. Data shows that optimal time for formalin fixation for most stains is 3-7 days.

After fixation, tissue can be stored for 1 to 3 days in 70% ethanol. Please consult resource staff if you need to store fixed tissue for a longer time.

Choice of Fixation:

Consult with us when planning experiments

The choice of a fixative depends on the goals of the study. Certain fixatives can result in superior morphology but give poor results when staining. Some protocols require that the tissue not be fixed at all. Please consult with us when planning experiments. If tissue is improperly fixed for a given technique, frequently no corrective action is possible. For example, certain fixatives disrupt the tertiary structure of the GFP molecule permanently inactivating the fluorescence.

Fixation is often an important consideration when preserving tissue for immunohistochemistry. Some antibodies cannot recognize antigens if they are cross-linked in a certain manner. There are some techniques, called antigen retrieval, which may be used to uncover antigens after fixation. However, these techniques do not work for all antigens.

Penetration:

The penetration of fixatives can be improved by opening up tissue.

Fixatives penetrate tissue at different rates. Non-coagulant fixatives, such as formalin, continue to cross-link proteins for as long as they are in contact with the tissue. Coagulant fixatives, such as alcohol and picric acid, achieve their full effect upon penetration as the protein coagulates.

The rate of penetration can be affected by heat but often not by concentration of fixative. As mentioned before, the penetration of fixatives can be improved by opening up tissue. In addition, the presence of fecal matter and stomach contents can inhibit the penetration of fixative, as well as damage tissue during sectioning. This material must be removed before fixation.

Tissue Storage:

Please consult Resource staff for the appropriate conditions for each fixative

Tissue storage is dependent on the type of fixative used. Please consult resource staff for the appropriate conditions for each fixative.

pH:

Between 7.2 to 7.4

The pH of a fixative can be very important for certain applications. In order to preserve fine ultrastructure, fixatives should be buffered to a pH of 7.2 to 7.4.

Osmolality:

Normal phosphate buffered saline (PBS) based fixative

If cells are fixed in a hypertonic solution, the cells may shrink. If the cells are fixed in a hypotonic solution, the cells may swell and burst. For that reason, we recommend using a normal phosphate buffered saline (PBS) based fixative.

Common questions about Formalin

How do I prepare formalin for tissue fixation?

10% Neutral Buffered Formalin (NBF)

Formaldehyde, 37% to 40% 100 ml
Distilled water 900 ml
Sodium phosphate, monobasic (NaH2POH20) 4 g
Sodium phosphate, dibasic (NaHPO4) 6.5 g
pH to 6.8

Can I order 10% NBF?

Yes! In fact we encourage it. We order our 10% NBF from VWR or Fisher Scientific.

VWR:

BDH0502-1LP: 1 liter bottle

BDH0502-4LP: 4 liter bottle

BDH0502-20L: 20 liter carboy

Fisher:

SF100-4: 4 liter bottle

SF100-20: 20 liter carboy

 

How long should I fix a sample in formalin?

Please consult Experimental Histopathology staff and “Points to consider regarding Tissue Fixation”.

Please consult the Experimental Histopathology Shared Resource staff if you have any questions regarding these points. As always, we encourage investigators to consult with us during the planning stages of an experiment. This allows us to provide the best possible service to you and ensure the highest quality tissue for your studies.

 

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Thomas Building, DE-360
(206) 667-6166
exphisto@fredhutch.org

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