[Histonet] Microtome calibration?

[Rittman, Barry R]

The microtome setting is the indication of how far the mechanism will advance with each stroke.
It cannot be an accurate measure of how thick a section will be cut as this depends on many other factors.
In general the thinner the section setting, the greater the possible variation.
One method that works is to stain a piece of fairly homogeneous tissue of known surface area that will accompany the specimen into the wax block.
Once the section has been cut you can remove the calibration piece of tissue (proteins also works) and dissolve. Then measure the amount of dye using  a spectrophotometer. This will give you the total volume of the calibration tissue and as you already known the cross sectional dimensions you can calculate the thickness of the section.
As an alternate can mount the section, carry out image analysis of the mounted stained calibration tissue for optical density using this calibration tissue.
Barry
 
From: histonet-bounces <@t> lists.utsouthwestern.edu on behalf of AGrobe2555 <@t> aol.com
Sent: Tue 5/22/2007 10:53 AM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] Microtome calibration?



Good morning,
A question was raised as to "how do you know the sections are  the
appropriate thickness?".  Does one trust the settings on the  microtome (Leitz 1512)? 
Is it an experienced eye that knows when they are  "right"?  Or is there some
instrument that will do this measurment?   Is there some way to calibrate the
microtome to ensure that the sections  actually are Xum?

Thanks,
Albert 

Albert C.  Grobe, PhD
International Heart Institute of Montana Foundation
Tissue  Engineering Lab, Saint Patrick  Hospital






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