Common Microtome Tissue Sectioning Troubleshooting and Tips
| January 16, 2023
Paraffin sectioning is the most common histological technique applied in preparing tissue sections. It does not only observe the form and structure of normal cells, but it is also the major pathological and forensic scientific method applied for studying, observing and determining the changes in the form of cellular tissues. It is also widely used in other fields of scientific research.
Living cells or tissues are mostly transparent and colourless. Due to the lack of image contrast between different tissues and cellular structures, it is difficult to differentiate between them under regular glass lens. Tissues die fast and decay after leaving the organism, losing its original structure. Therefore, in order to get a clear image of their forms and structures, tissues have to be fixated, embedded, cut and dyed in preparation of sample sections.
Paraffin sectioning involves steps including sampling, fixation, rinsing and dehydration, waxing, embedding, cutting and mounting, dewaxing, staining, dehydration, clearing, mounting and so on. Cutting is one of the most crucial parts that determines the section results. There’re lots of factors affecting the microtome tissue sectioning performance. What are the common problems of microtome tissue sectioning? How to solve these problems?
(1) Uneven sharpness of the blade results in discrepancy. Move the blade to get a sharper edge. (2) It is found that the blade has not fully covered the paraffin block. Adjust the blade position until it can cut out a complete block. (3) If the hardness of the paraffin block is uneven on both sides, try to use the sharper edge of the blade to reduce friction. (4) If the tissue is not in the central position of the paraffin block, use the blade to cut out some of the paraffin wax to move the tissue back to the centre or re-embed it.
No formation of sections due to separation of ribbon
(1) If the air is too dry, use a humidifier or breathe on the sections. (2) Is the retraction function of the microtome sectioning machine switched off? Switch it on. (3) Too little residual paraffin on block edges? Re-embed and try to make sure the tissue is in the centre. (4) Is the blade angle inappropriate? Adjust the clearance angle and bevel angle.
(1) Is the paraffin block too cold? Increase the temperature accordingly. Use a brush to spread out the section and press it firmly while cutting it into 2 to 3 flattened slices. (2) Paraffin block is too hard? Add softer wax. (3) The blade is too blunt? Change the blade. (4) The bevel angle is too big? Narrow it down.
Ribbons crumble and stick on the knife
(1) Is the temperature of the block too high? Lower the temperature appropriately. s the paraffin block too soft? Freeze it more. (2) A thin layer of paraffin remains on top of the blade? Clean the blade and scrub the excessive paraffin wax on top. Use Xylene to clean the blade. (3) Blunt blade? Get a new blade. (4) Too many fats? Remove the fats during preparation and squeeze out the fats at the stage of embedding.
(1) The knife is chipped? Move the blade. (2) Impurities in paraffin wax? Remove the impurities and get high-quality paraffin. (3) Excess wax remains on the blade? Clean the blade. (4) The tissue is too hard? Soak it in the water to soften it. (5) Hemostats inside the tissue? Take them out.
Train lines on Sections
(1) Are the clamps and other parts of the machine getting loose? Make sure everything is firmly locked. (2) The blade bevel angle is too wide? Narrow it down to an appropriate degree. (3) The specimen extends too much? Retract it appropriately and move the knife holder forward. (4) The paraffin block is not firmly locked? Clean the paraffin block and the cassette clamp. (5) The tissue is too hard? Decalcify its surface and cut it slowly. (6) Are there worn microtome parts? Maintenance or machine replacement is necessary.
(1) Are there worn microtome parts? Maintenance or machine replacement is necessary. (2) Improper way of exerting force on the machine? Adjust your physical strength. (3) Are the clamps and other parts of the machine getting loose? Make sure everything is firmly locked. (4) Is the paraffin block too hard? Soak it in the water. (5) Is there any temperature change? Refreeze the paraffin block.
Tissues crack and break
(1) Improper dehydration? Dehydrate it again. (2) Is there any clearing agent residue? Increase the time for infiltration and then re-embed the sample. (3) Brisky sample under the influence of clearing agent and dehydration machine? Adjust the dehydrating process. (4) The tissue sample is too big or too hard? Decalcify and embed it separately.