[Histonet] Sectioning in sliding microtome issues

[Maria Mejia]

Hello Maria,

Well, it's interesting that the Thermo HM 450 sliding microtome, looks very much like the Microm HM450 sliding microtome
that I used (A LOT) at UCSF.  You must be cutting (30um or 40um??) free-floating sections.
Questions:
> How thick (mm) are the frozen brain block?
> AND, are you cutting a whole brain from a non-human primate?
> Are you using a solid brass freezing stage - mounted to your sliding microtome?  The type with 2 small wells (1 in the front 
   & 1 in the back). 

If you're cutting a (whole) brain block - large or small, I would strongly recommend the use of Cryogel - which is the bomb! 
I've used this stuff to cut excellent sections from 1mm to 9mm thick frozen brain blocks.  Switching to cryogel, will significantly 
reduce your rate of loosing chunks of tissue at the end of the block because it supports like no other freezing resin. When you
use cryogel or OCT or any other freezing media, make sure there are NO air bubbles in the supporting media.

I would also check all the knobs on your sliding microtome & freezing stage - make sure that nothing is loose!  Equally important, 
is maintaining consistent freezing temperature of the frozen brain block & of the supporting resin during sectioning. 
Especially since the sliding microtome is not in a cold chamber like a cryostat.  So, if your cutting room is too warm - this warm environment 
will dramatically affect your freezing temperature of tissue block & resin AND ultimately your cutting.

For this reason, I chose to use the Physitemp freezing stage - the freezing stages come in 3 sizes and at UCSF we used the larger one 
- the BFS-30MP (4x4 inches square).  Here is the link;  http://www.physitemp.com/products/FreezingMicroTomes/bfs-mp.html
You'll need a water tank - link:  http://www.physitemp.com/products/accessories/ptu3.html

If you would like my freezing protocol, just let me know.

Regards
Maria Mejia
Affymetrix, Inc








 

  



On Apr 3, 2013, at 6:06 PM, Maria De Los Angeles Navas wrote:

> Hello All,
> 
> We section fixed frozen brain that we mount on the microtome using either 30% sucrose or OCT and we keep the temperature down by surrounding the tissue with crushed dry ice constantly (every 10 minutes).
> 
> One of the problems I have had, and also the person who trained me also had, is that when getting towards the end of the block a big chunk of the tissue gets lose and ruins the sectioning of that last part. Any tricks, tips to make this better?
> 
> Also, this week the microtome has been skipping one section all together and cutting the next one twice as thick. We have a Thermo HM 450 microtome and I was wondering if people have experienced this problem with this same microtome (or others) and if you know what the problem could be and how to go around it. I don't think is temperature, while testing the microtome today I was being extra careful by keeping the temp constant, but you never know for sure.
> 
> 
> Thanks sooo much,
> 
> Maria
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