HREM SAMPLE PREPARATION

Dehydration and infiltration

In preparation for embedding and imaging, samples are dehydrated then infiltrated with a JB-4 dye mix. The exact procedure depends on the size and tissue density of the sample. The procedure below is intended as a guide, but the parameters are empirical and some experimentation may be required with your own samples.

Consumables

JB-4 dye mix preparation

Final volume (ml)80100150200300
Solution A (ml)80100150200300
Catalyst (g)1.001.251.882.503.75
Eosin B (g)0.2200.2750.4130.5500.825
Acridine Orange (g)0.0450.0560.0840.1130.169
  1. Measure Solution A and place on a stirrer.
  2. While mixing vigorously, add the catalyst slowly to avoid the formation of lumps.
  3. Slowly add both dyes.
  4. Stir for at least 4 hours (preferably overnight) at room temperature or below.
  5. Filter the mix through 0.22 μm PES membrane to remove dust and any undissolved dye.
    • There should be very little or no undissolved dye to remove. If there are lots of undissolved dye grains you may need to start again. The mix will break standard filter membranes.

At this point, the JB-4 dye mix can be stored at 4 ℃ for 2-3 weeks. Any leftover mix can be used to prepare 50:50 JB-4/methanol for infiltration.

Procedure

For an E14.5 mouse embryo (roughly 10mm by 5mm):

Dehydration

  1. Fix the sample overnight in Bouin's fixative, then transfer to PBS.
    • Other fixatives including 10 % formalin, 4 % paraformaldehyde and Dent's can be used. We prefer Bouin's fixative for whole embryos since it gives better preservation of the structure of soft mesenchymal tissue, which tends to collapse during dehydration.
  2. Over the course of 1-2 days, wash samples with repeated changes of PBS for 2 hours per wash.
  3. Dehydrate samples using a MeOH series (10 %, 20 %, 30 %, 40% 50 %, 60 %, 70 %) for 2 hours per step.
  4. If using Bouin's fixative, wash the sample in 70 % MeOH containing 1% ammonia for three minutes.
    • This helps remove any residual yellow picric acid staining left from the Bouin's fix.
  5. Complete dehydration using MeOH mixes (80 %, 90 %, 95 %, 100 %) for 2 hours each. Samples can be held overnight at any stage prior to 90 % if necessary.

Infiltration

  1. Immerse the sample overnight in a 50:50 mix of MeOH:JB-4 dye mix.
  2. Briefly rinse in JB-4 dye mix to help minimise residual MeOH, then immerse in several ml of fresh JB-4 dye mix.
  3. Leave to infiltrate at 4 ℃, preferably with gentle rocking.
    • For example, infiltration of an E14.5 embryo usually takes 3 or 4 nights.
  4. The sample is now ready to be embedded.

For small embryos or embryo tissue pieces, the length of dehydration and infiltration steps can be reduced drastically:

  • For E9.5 embryos, dehydration steps need only be 30-60 minutes and infiltration takes only 3-4 hours.
  • For denser embryo tissue such as an E14.5-E18.5 heart, we dehydrate in 1-hour steps and infiltrate overnight.
  • For larger or much more dense samples such as adult tissues, successful infiltration can take more than a week and needs to be determined empirically.

NEXT: Embedding

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