[Histonet] Issue with Technovit 7100 - nerve samples

[Chaitanya Kolluru]

Hi,

I'm working on a protocol to embed human nerve samples (about 3 mm in
diameter, 5-7 mm in length) in glycol methacrylate and cut 2-3 micron thick
sections.

The sectioning seems fine at the start, but as I section deeper into the
sample, I find a lot of gaps forming in between the fascicles, which I'm
assuming are air bubbles.

I'm attaching an image of the block surface after sectioning about 500
microns into the sample. I'm also attaching a text file of my protocol if
that can help troubleshoot the issue.

Any help on how I might be able to reduce this artifact would be much
appreciated. If an alternative resin would alleviate this issue, please do
let me know.

Thanks in advance,
Chaitanya

Link to image of the block surface:
https://cwru.box.com/s/5dsd7ngazlhf0jxga4apqh47a75uzb84
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1. Samples were extracted from cadavers and left in formalin for��more than a couple of month.
2. Cut 5 mm long nerve segments and rinse in PBS for 1h. Post-fix nerve in 2% osmium tetroxide for 2 hours.
3. Perform gradual dehydration in increasing steps of ethanol, about 2 steps per day for a total of 3 days (with rotation).
4. Pre-infiltrate specimen with a 1:1 mixture of 96% ethanol and 100% GMA solution for 2 days.��Perform vacuum cycles for about 30 min everyday with 35 psi in a vacuum pump.
5. Immerse sample in the infiltration mix (1g Hardener 1 in 100 ml GMA) for a total of 6 days, with 2 changes of fresh infiltration mix in between. Same vacuuming protocol as previous.
6. Polymerize with 15 parts infiltration mix and 1 part Hardener 2.
7. Polymerize in Teflon molds in vacuum at room temperature (25 deg C) for a couple of hours, followed by polymerization in the oven at 37 deg C. I see that the blocks are usually soft after this, so I let the samples continue to sit in the mold in the oven at 55 deg C for 48 hours.
8. Remove from mold with Technovit 3040, clamp on the motorized microtome (Microm) and section with tungsten carbide knife at 13 degrees clearance angle with a slow speed setting at 2-5 micron section thickness.