[Histonet] Using LR white with a tungsten carbide knife

[Chaitanya Kolluru via Histonet]

Hi,

I’m looking for some advice on using LR white on an osmium stained tissue.
Unfortunately, I don’t have access to an ultra-microtome or diamond knives.

Has someone here tried sectioning LR white with tungsten carbide knives on
a motorized rotary microtome? Are there any sectioning artifacts that I
might see? Also, LR white seems to be available in three grades  - soft,
medium and hard. Is the soft grade recommended with my setup?

Thank you,
Chaitanya
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[Histonet] Issue with Technovit 7100 - nerve samples

[Chaitanya Kolluru via Histonet]

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
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.___
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[Histonet] Looking for a sliding microtome

[Chaitanya Kolluru via Histonet]

Hi,

I'm searching online for a microtome where the sample is fixed and the
knife is moving. The microtome cutting movement needs to be motorized and
automatic.

Is there a microtome that has this functionality or is this something that
simply does not exist?

Thank you!
-- 
Chaitanya
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