If you’re involved in biological research, chances are at some stage you’ve submitted tissue specimens to a histology lab. Somehow they magically produced beautiful slides for you – each containing thin sections of your specimens, ready for microscopic evaluation.
Have you ever wondered how the histology technician does this? Read on for the five important stages in histology slide production:
1. Tissue fixation
Slide preparation begins with fixation of your tissue specimen. This is a crucial step in tissue preparation, and its purpose is to prevent tissue autolysis and putrefaction. For best results, your biological tissue samples should be transferred into fixative immediately after collection. Although there are many types of fixative, most specimens are fixed in 10% neutral buffered formalin. The optimum formalin-to-specimen volume ratio should be at least 10:1 (e.g., 10ml of formalin per 1cm3 of tissue). This will allow most tissues to become adequately fixed within 24-48 hours. Formalin containers should be capped and leak-proof, and labelled correctly.
2. Specimen transfer to cassettes
After fixation, specimens are trimmed using a scalpel to enable them to fit into an appropriately labelled tissue cassette. Specimens should not be so big that they fill the cassette – they are trimmed so as not to touch the edges. Additionally, they must not be too thick (ideally they should be less than 4mm), otherwise they risk being “waffled” when the cassette lid is closed. The filled tissue cassettes are then stored in formalin until processing begins.
3. Tissue processing
Processing tissues into thin microscopic sections is usually done using a paraffin block, as follows:
- Dehydration is the first step, which involves immersing your specimen in increasing concentrations of alcohol to remove the water and formalin from the tissue.
- Clearing is the next step, in which an organic solvent such as xylene is used to remove the alcohol and allow infiltration with paraffin wax.
- Embedding is the final step, where specimens are infiltrated with the embedding agent – usually paraffin wax. The tissue becomes surrounded by a large block of molten paraffin wax, creating what is now referred to as the “block”. Once the block solidifies, it provides a support matrix that allows very thin sectioning.
Your tissue specimen is now ready to be cut into sections that can be placed on a slide. Wax is removed from the surface of the block to expose the tissue. Blocks are chilled on a refrigerated plate or ice tray for 10 minutes before sectioning. A microtome is used to slice extremely thin tissue sections off the block in the form of a ribbon. The microtome can be pre-set to cut at different thicknesses, but most tissues are cut at around 5 µm. Check out this article for more ways to slice tissue sections.
Once cut, the tissue ribbons are carefully transferred to a warm water bath. Here they are allowed to float on the surface, and can then be scooped up onto a slide placed under the water level. Charged slides work best for this process – they improve tissue adhesion to the glass, and help to reduce the chance of sections washing off the slide during staining. Slides should be clearly labelled, and then allowed to dry upright at 37oC for a few hours to gently melt the excess paraffin wax, leaving the tissue section intact.
Most cells are transparent, and appear almost colourless when unstained. Histochemical stains (typically haematoxylin and eosin) are therefore used to provide contrast to tissue sections, making tissue structures more visible and easier to evaluate. Following staining, a cover slip is mounted over the tissue specimen on the slide, using optical grade glue, to help protect the specimen.
So as you can see, slide processing is no breeze – it’s quite an intricate work of art! Although you may want to learn how to do this to help cut costs in the lab, I’d advise you to think twice about this, and instead send the specimens to a histology laboratory for this purpose – especially if tissue evaluation is an important part of your study. You’ll save yourself a lot of stress and time by leaving this job to an experienced histology technician, and your slides will be much easier to evaluate.
Do you prefer to make your own slides, or send tissues to a histology lab for processing?