[Histonet] chatter in fly head paraffin section

From:Zhenming Yu

Dear Histonetters,

I'm looking for some guidance in obtaining good paraffin sections from
fruitfly brain/eye. We repeated saw chatter in the brain region and are 
desperately trying to troubleshoot the problem.

We have tried 10% NBF, 4% paraformaldehyde, Bourin's for fixation. 

And for tissue processing, we used the following schedule: 

70% Alcohol 3 hours
95% Alcohol 2 hours 
100% Alcohol 2 hours 
100% Alcohol 2 hours   
100% Alcohol 1 hours 
Xylene 1 hour 
Xylene 1 hour 
Xylene 1 hour
Paraffin(60C): 2 hours
Paraffin(60C): 2 hours---we didn't use vacuum at this step considering fly 
heads are quite small. Could this cause inadequate praffin infiltration and 
chatter in sections?

The technician did leave paraffin in the processor melting at 60C for long 
period of time--she never shut off the power after the cycle is done. She also 
always left the embedding station on with paraffin melting at 62C. Could this 
cause deterioration of paraffin?

For sectioning, we've tried using brand new blade, adjusting clearance angle, 
slowing down cutting speed etc. None seems to help. 
Any suggestion would be greatly appreciated. Thank you all. 

Zhenming Yu 
Univ. of Penn
Philadelphia, PA

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