HREM FAQ

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How long can I use JB4/dye mix?
The manufacturer's advice for JB4 is to use it within a couple of weeks and addition of dyes doesn't seem to affect this. We have not tested older mix. Any leftover old mix can be used for preparing 50:50 JB4/methanol or rinsing samples between the JB4/methanol step and infiltration with current JB4/dye mix.
Why is my JB4 polymerisation so slow?
Normal polymerisation of JB4/dye mix taken directly from the fridge should begin within approximately 15 to 30 minutes. The mixture will begin to become viscous and if there is any surplus you should feel it begin to warm up. If the mix is taking substantially longer or does not polymerise fully at all check your components by testing polymerisation of JB4 without dyes. Incomplete removal of methanol during sample infiltration appears to inhibit polymerisation.
Why are my JB4 blocks soft?
If the blocks are very soft and jelly-like or rubbery immediately after removal from the mould, there is likely to be something wrong with your JB4 mix or its components. Normal blocks will sometimes feel slightly “springy” when squeezed, especially if they are large in size. This can be cured by baking the blocks at 90 to 100 ℃ for several hours (conveniently, overnight). Blocks that have previously polymerised hard (or have been baked hard) can become slightly soft during prolonged storage, either at room temperature or in the cold. We are not sure what causes this and in the UK, this seems to be more of a problem in the summer months. It can be solved by rebaking the blocks.
Why do my JB4 blocks break off their plastic chuck?
This can happen when there is insufficient JB4 polymerised through the central hole and the crosshair gaps in the base of the chuck. Add a little fresh polymerisation mix to top up the base. If your sample has broken from the base, use abrasive paper to ensure the attachment surface of a fresh base is completely flat (- they are NOT flat from manufacture); do the same with the base surface of the JB4 block and then use SuperGlue contact adhesive to reattach to the plastic chuck.
How do I mend a broken JB4 block?
Blocks can occasionally break during sectioning or trimming. Excessive baking can result in brittle blocks and these can sometimes shatter, especially if the section depth is unusually large. SuperGlue is very effective for repairing broken blocks.
Can I trim JB4 blocks?
Yes. Bake the block for 5 to 10 minutes at 90 to 100 ℃ and it will become soft and pliable. It will remain like this for about 1 to 2 minutes after removal from the oven. During this time, it is possible to cut the block by firmly pushing a single side razor blade (or utility knife blade) through the block with a slight sawing motion. Be quick, as this rapidly becomes impossible as the block cools down and hardens!
Why do my images have a very bright region in the centre of the tissue?
This is due to insufficient time of infiltration. The period is important not only for ensuring complete penetration by the methacrylate, but also for infiltration and tissue staining by the dyes. We do not fully understand why, but the latter process seems to be important for tissue visualisation and can take longer than the former. As a result, particularly dense tissue can show a very bright region in its centre with little or no imaging of the tissue. If this happens, you need to increase the infiltration period. (Note that prolonged infiltration times will also darken the overall grayscale range of the tissue in the image).
Why do my images have scratch lines?
Straight scratch lines perpendicular to the microtome blade are caused by damage to the edge of the blade. These accumulate with use of the blade and are sometimes present even with a new blade. For optimal quality images and minimal scratch lines, we recommend using a new portion of blade for each sample.
Why do my images have ripple-like wavy lines?
Irregular, wavy lines oriented roughly along the axis of the cutting blade are caused by soft JB4. This tends to happen when sectioning nearer the base of the block, probably because this region is more likely to suffer from inhibition of polymerisation by oxygen.

If you have any other questions about HREM, please email contact@dmdd.org.uk.