Penn Center for Musculoskeletal Disorders
Tips and Tricks for Better Histology Results
Ensure adequate fixation and decalcification
Tissue that is not completely fixed or decalcified will not section well. Commonly used fixation and decalcification protocols are available here. If you are not sure of the correct fixation or decalcification technique for a specific tissue type, please consult with Dr Tang. For blocks that retain mineral deposits, it may be possible to decalcify the exposed block surface prior to sectioning.
Ensure adequate dehydration and paraffin infiltration during tissue processing
Optimal processing is critical to obtaining high quality sections. Since paraffin is immiscible with water, the main constituent of tissue, samples need to be dehydrated by progressively through more concentrated ethanol baths. This is followed by a clearing agent, usually xylene, to remove the ethanol. Finally, molten paraffin wax infiltrates the sample and replaces the xylene. Processing under vacuum improves infiltration and decreases processing times. Processing times need to be optimized based on tissue size, type and application. If you are unsure of the correct processing cycle for your sample, please consult with Dr Tang.
Chill your paraffin-embedded tissue blocks on ice
To promote good ribbon generation, the paraffin block needs to be sufficiently hard to enable the sections to be cut on the microtome. This is routinely achieved by placing the paraffin blocks on ice, cooling both the tissue and the wax to a similar consistency and swelling the tissue to make it easier to section. As the block heats up, however, the paraffin softens and expands, resulting in corrugation and variations in the consistency of the sections, rendering some unsuitable for use. It is not recommended that blocks be placed in the freezer since the paraffin and tissue can crack. Freezing blocks will introduce artifacts to tissue sections.
Make sure the water bath is set at the correct temperature
If the water is too cold, sections may not spread well. Alternatively, if the water is too hot, the paraffin will melt. The ideal water bath temperature is 38-42ºC.
Check the ambient environment
While it may not always be possible to tightly control, particular in an open and shared lab space, the ambient environment can sometimes impact sectioning quality. A room that is not too cold, hot, humid, or drafty (about 24 to 26 C) is best. If you believe the ambient environment is unsuitable, please consult with Dr Tang so that it can be rectified with facilities.
How do I reduce wrinkles when cutting plastic sections?
Plastic sections with some wrinkles is a very common problem, and one that results in poor imaging quality at low magnifications. Here are some useful tips to minimize wrinkles:
- Thicker sections will have more wrinkles. Try cutting sections as thin as possible (4 to 6 µm), as this will be much easier to keep flat when placed into the alcohol.
- Leave the sections in 70% ethanol for 7 to 10 minutes compared to the usual 3 minutes.
- Press the section using your finger under the plastic film evenly and for several seconds.
- Roll sections lightly and carefully 3 to 5 times on the slide.
How do I stop plastic sections from falling off during staining?
Plastic sections have 20-30% possibility of falling off the slides during routine staining, even though you have used slides coated with the 2% gelatin. Is there anything we can do now to stop or reduce the sections coming off? Here are some useful tips to prevent this from happening:
- Use a higher concentration of gelatin (3-4%) to coat your slides.
- Reducing the washing time, especially for the alcohol solutions.
- Don't start staining immediately following removal from baking in the 24°C oven. Leave them at room temperature for a few days.
- Be sure to transfer the slides very gently between staining steps.