[Histonet] Vibratome skipping sections

[Andrew Gillis]

Hello,

I have just started using a vibratome (Leica VT1000S) to cut sections of 
fish embryos after whole mount in situ hybridization, but I've 
encountered a problem with this. I have been setting the vibratome to 
cut sections of 50um to 70um in thickness (on the recommendation of a 
colleague who routinely uses this instrument), and I've noticed that 
after a few nice sections, the machine starts skipping a section (i.e. 
the blade will just pass over the block). When this happens, the next 
section to be cut is approximately twice as thick (i.e. if my section 
thickness is set to 70um, the blade will miss a section, and will then 
cut a section that is approxaimtely 140um thick).

I'm not sure if this is a problem with the settings I'm using, or with 
the way I prepare the blocks.... I tend to cut with a speed of 5-7, and 
a frequency of ~8 (again, based on settings recommended by a colleague). 
To prepare the block, I embed the embryos in 30% gelatin in PBS, and 
once set, I trim it and fix the block in 4% PFA for 48hrs. I try not to 
make my blocks too tall, and they seem nice and firm when I mount them 
on the machine....

There are certainly a lot of variables that I could play with here - and 
I could probably spend weeks fiddling with it! I would really appreciate 
hearing from anyone else has experienced this, and who has ideas of how 
to fix it.

Thank you very much,
Andrew