SV: [Histonet] chatter in fly head paraffin section

[Eva Alströmer]

If you use 4% buffered NBF be sure that you have washed out the salt
crystals from the 4% buffered NBF as this cristals can cause scratehes
in cutting.

Best regards,
 
Eva Alstromer

-----Ursprungligt meddelande-----
Från: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] För Zhenming Yu
Skickat: den 20 juli 2005 14:50
Till: histonet <@t> lists.utsouthwestern.edu
Ämne: [Histonet] chatter in fly head paraffin section

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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