[Histonet] Experiencing Chatter in paraffin ribbons

[Nicole C Walsh]

Hi,

We routinely cut 5micron paraffin sections of decalcified mouse bones  
including whole hindpaws. We use Surgipath Infiltration medium for  
infiltration steps and then Surgipath EM-400 Embedding paraffin for  
embedding. This has worked well in the past, but since moving to a  
new institution and using different microtome setup we are now having  
problems with sectioning.

Particularly we are experiencing a lot of chatter in the surrounding  
wax. We routinely keep the blocks cold when sectioning.. cooling in  
between each  ribbon of 5-7 sections (maintained at 4degC on cooling  
block on embedding station, with slight coating of block softener  
(60% glycerol, 20% ethanol and 20% water)). The tissue generally  
comes off intact suggesting that infiltration of the tissue is  
reasonable.

We are trying to eliminate possible reasons for our problems.

A couple of things have changed since moving here..

We recently refreshed our paraffin supplies and this seems to have  
been when our troubles really started. But Surgipath have said that  
they have not changed the formula, but have given us paraffin from a  
different lot number to try.

We've had a maintenance rep recently service the microtome (Microm  
HM315)  and he could not find reason for the chatter (we section with  
angle set to 10 as this is what the rep suggested should be used for  
this microtome, and have tried varying the angle previously with no  
success)

We used to use surgipath high profile blades (non-coated) with  
success (and I've seen discussion on histonet to say that the high  
profile blades are better for hard tissue).
But the microtome that we now use will only take low-profile blades  
and so far we've tried the Richard Allan Scientific low profile  
blades (found that these became blunt quickly) and are now trying the  
teflon coated low profile blades from surgipath.

If anyone has any suggestions for things that we might try that could  
help correct our problems we would be very grateful.

Thanks in advance,

Nicole