Methods and Techniques Discussion > General Histology

50um thick Paraffin Sectioning

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DMSVSU:
Our lab is currently working on increasing our sectioning methods from 10 micron sections to 50 micron sections. We are using rat brains (sagittal sections) embedded in paraffin wax, and an automatic microtome (model Leica RM225).  We have been basing our techniques off the protocol at http://www.ihcworld.com/_protocols/histology/gelatin_slides.htm
but I had a few questions concerning the actual slicing procedures used in the description. 

I was wondering if anybody has been able to slice continuous ribbons of tissue 50um thick?  At the moment, each section will instantly curl as it goes across the automated microtome blade.  The only way to get a proper section is to turn the speed down and manually follow the tissue with a paintbrush as it comes into contact with the blade.  To anybody who has used an automated microtome you would recognize that this is not an easy thing to consistently do without brushing the bristles against the blade itself, or worse...getting it caught between the tissue sample and the blade. 

We have been trying to keep the protocol as consistent as possible, (trimmed tissue samples, 45* C water bath, tissue samples at room temp, etc). 

Does anybody have any suggestions as how to prevent section curling, or how to get a ribbon of tissue to consistently be developed as we go along?
It's manageable going one section at a time, but it would be nice to find a better way of preventing curling in order to save tissue within each sample. 

Thanks in advance.

excalibur:
50um sections rarely ribbon. You will need to use a brush to hold each section down as it comes off the blade to keep it from curling. Use another damp brush or fine forceps to pick the section up for transfer to the slide or waterbath.

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