PREPARING BLOCKS FOR IMAGING

After embedding, polymerised blocks are removed from moulds and trimmed. We recommend samples are baked to ensure optimal hardness. This stage is optional, but it can improve the stability of the sample during sectioning. Baking times are empirical and some experimentation may be required with your own samples

Procedure

  1. Invert the mould onto paper towels to remove mineral oil and unpolymerised JB-4 mix.
  2. Check that polymerised JB-4 is visible in both the central hole of the chuck and through the base of the chuck. (Image 1)
    • This is essential to avoid the sample block breaking off during sectioning. If necessary, a little fresh JB-4 embedding mix can be added to the top of the chucks and left to polymerise for a few hours. If the block does break off, use abrasive paper to ensure the surface of the chuck is completely flat (it is not flat from manufacture). Do the same with the base surface of the JB-4 block and then use SuperGlue contact adhesive to reattach it to the plastic chuck.
  3. Remove blocks from the mould and wipe dry.
    • For the small (5 mm) EMS flexible moulds, blocks can be popped out by firm pressure from underneath. Excess polymerised JB-4 must be removed from the side of the chuck using a blunt blade to enable the block to fit in the HREM block holder. (Image 2)
    • For the larger PTFE moulds, grip the plastic chuck with an adjustable wrench and pull each block vertically. Be careful not to apply too much pressure with the wrench - this can cause distortion of the chuck, weakening its attachment to the sample block.
    • 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 JB-4 mix or its components. Normal blocks will sometimes feel slightly springy when removed from the mould, especially if they are large in size. This is usually fixed by baking.
  4. Bake the samples at 95 to 100 ℃ to ensure the blocks are completely hard before sectioning.
    • For small (5 mm) samples, 12 hours is sufficient. Larger blocks benefit from longer times. For E14.5 mouse embryos (14-16 mm depth blocks), we bake for 24 to 48 hours. The blocks will become soft and pliable after 5 minutes of baking, but harden rapidly after removal from the oven. Baking has no effect on the sample or subsequent image quality, but baking for excessive periods can produce brittle blocks that can be prone to accidental chipping or cracking.
  5. Cool the block in a fridge for several hours until completely hard.

It can be convenient to trim off much of the JB-4 cushion prior to HREM imaging. This can be carried out either on the HREM apparatus or using a manual microtome. Trimming can be performed either before or after baking and cooling of blocks. To measure the approximate cushion thickness, view the block on its side and illuminated from below in a bright light beam.

Occasionally, blocks can break during 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.

NEXT: Imaging

If you would like further advice on baking samples for HREM imaging, please email contact@dmdd.org.uk.

Image 1

Polymerised JB-4 is visible in the central hole and through the base of the chuck.

Image 2

Before (left) and after (right) trimming excess polymerised JB-4.