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An Introduction To Fixation For Histology: Think Before You Fix!

Image of a mechanic thinking before fixing a car to represent thinking before performing histology fixation

For many cell or tissue-based experiments, histology microscopy is likely to be the endpoint. In this series of articles, we want to illustrate how some joined-up thinking regarding the various stages of tissue or cell collection, fixation, and processing, subsequent staining, microscopic imaging, and analysis will produce better results.

In this article, we focus on the critical step of histology fixation.

The Aim of Histology Fixation

The aim of fixation is to preserve cells or tissues in as near a life-like condition as possible, prevent autolysis and putrefaction, and protect the tissue from damage during subsequent processing.  Fixatives have different actions: for example, crosslinking, precipitative, coagulative. They also have different penetration rates, usually measured in depth penetrated per mm per hour.

I normally recommend fixing in 20x the volume of fixative to tissue and fix for consistent periods of time. For example, I would normally fix samples 0.5 cm thick in at least one dimension for 24 hours in Neutral Buffered Formalin, or for 6 hours in Bouin’s solution.

I would then transfer samples into 70% ethanol (the first stage in many routine paraffin processing protocols) prior to processing.

Why Histology Fixation Choice is Important

Not only is the correct fixation of cells and tissues critical to preserving cells or tissues in as near a life-like condition as possible, but it is also important to ensure staining or fluorescence is of a good intensity for imaging.

Intense well-stained fluorescent cells or tissues will make imaging much easier whether you are using widefield, confocal, or super-resolution microscopy. Good staining will be achieved by using appropriate detection systems and suitably fixed cells or tissues.

10 Things to Consider Before Performing Histology Fixation

Now we know why histology fixation is important, let’s look at what you need to consider before fixing your tissues.

1. Choose the Right Temperature

Temperature is an important consideration in histology fixation: a low temperature will reduce autolysis in tissues, but lead to a slower penetration rate, so choose what is important to you.

I normally fix at room temperature with exceptions for tissue to be processed to resin or for some sensitive antigens where low-temperature fixation is recommended.

2. Think About the End Point

The choice of fixative is determined by the endpoint. For example, if an extremely high degree of morphological preservation is required for light or electron microscopy on resin sections, then the likelihood is that a glutaraldehyde-based fixative (perhaps in combination with paraformaldehyde) would be the method of choice.

As neither of these is effective at preserving lipid (which would be lost in the subsequent processing to resin or plastic) the tissue would need to be post-fixed in osmium tetroxide.

3. Understand the Downsides of Your Fixative

It’s important to know the negatives about your fixative so that you can make an informed decision before applying it to your precious samples.

Glutaraldehyde, for example, penetrates tissue slowly, meaning that tissue pieces need to be much smaller or fixation times much longer.

However, like many things in life, there is a trade-off for obtaining this high degree of morphological preservation. Generally, it is much more challenging to perform empirical staining techniques (such as Haematoxylin and Eosin, PAS, etc.) or molecular tissue probing techniques using antibodies (e.g., immunohistochemistry) or nucleic acid probes [e.g. in situ hybridization (ISH)].

Learning what common histology fixatives do to your samples is one way to understand both the positives and negatives of your fixative.

4. Consider the Cryostat

The other end of the scale is to use frozen (or cryostat) sections with no or minimal fixation. For example, a frozen section can be produced air-dried onto a slide and directly stained.

Therefore, it’s not had the chemical exposure of fixatives or the alcohols, solvents, and the heat which is associated with paraffin processing.

For certain techniques, this has huge advantages: for example, in the demonstration of lipids, immunohistochemistry, and immunofluorescence for certain antigens, and for brain tissue where it can give excellent results.  However, the generally poorer morphology obtained for other tissues overall makes this choice less mainstream.

5. If in Doubt, Walk the Middle Ground

Like our politicians, most of us try to appeal to the middle ground when it comes to fixation! For the time being, the middle ground in fixation is formalin-fixed paraffin-embedded (FFPE) tissue.

It is used extensively in diagnostic pathology. Pathologists are familiar with the chromatin pattern of tissues fixed in this way and it provides a good degree of morphological preservation and chemical preservation such that proteins are well preserved, although often masked (which can be undone) and mRNA and DNA can also be hybridized.

6. Be Aware That Different Disciplines Have Different Preferences

Antibody manufacturers often give conditions for use on FFPE tissues, probably because they have optimized and validated these reagents on this easily available type of sample. But like political parties, different disciplines have different preferences!  For example, for studying macrophages in one of our research centers, Methacarn is the fixative of choice.

7. There is No One Solution to Fix Them All

As the title says: “think before you fix”.  It doesn’t mean 4% Neutral Buffered Formaldehyde is the best fixative for every application, it could in fact be detrimental to obtaining the best results in an antibody-based experiment, or indeed morphological preservation.

8. Tissue Dictates Histology Fixation

For example, in the early 90’s I used to study spermatogenesis and therefore worked on the testis. The testis is a fairly fluid-filled organ with a heterogeneous population of cells contained within two main compartments, the tubules, and interstitium.

In the rat, there are 14 stages of spermatogenesis so that in any cross-section of testis the seminiferous tubule could be characterized as one of 14 stages. These stages are distinguished by subtle differences in the morphological appearance of different cell types. In the testis, I wanted to look at mRNA expression using ISH. Most literature recommended 4% paraformaldehyde for ISH.

9. Try Different Fixations

So which fixative did I choose?

The answer is, of course, we chose, well………several! At least for the initial evaluation!

The reason was that we could see from the published literature that paraformaldehyde would not give the degree of morphological preservation needed to recognize the subtle differences in the cellular population of the testis.

We might get an mRNA signal, but if we could not identify precisely the cell type and stage of spermatogenesis, then the information obtained would be of little value. So we chose several and, in the end, selected Bouin’s solution, which although it gave a slightly less sensitive signal by ISH, gave the morphological preservation needed to identify at which stage of spermatogenesis the gene was expressed.

Perhaps I was lucky because these 20-year-old samples can still be used for ISH today and it has proven in my hands to be an extremely good fixative for immunohistochemistry.

Would I recommend you fix everything in Bouin’s solution? Of course not! It was the ideal fixative for our tissue and our desired endpoint, but it does not mean it is ideal for every application. Finding your ideal fixative might involve trying different ways of fixing your samples, preparing them in different ways, and applying different staining strategies.

For most applications, sticking to the middle ground, certainly initially, is a good strategy for fixation but if it is either not giving you the endpoint that you need, or is just plain not working, then keep an open mind and explore different fixation strategies to obtain the best results.  Do this near the start of a project where you have the flexibility to change direction.

10. Be Consistent

And finally, once you have made your fixation choices (e.g. fixative, time, and temperature) then keep it consistent and, who knows, you could still be using these samples in 20 years’ time!

Want to know more about histology? Visit the Bitesize Bio Histology Hub for tips and trick for all your histology experiments.

Originally published July 17, 2012. Reviewed and updated August 2021.

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