[Histonet] dehydration time for a relatively large sample

[Rui TAHARA]

Hi, 

Thanks for prompt response. 
We unfortunately do not have a processor in our lab at university..The protocol i wrote was working in quail embryonic samples (just before hatching). I process the tissue manually and cannot process the tissue with strict time schedule. Thus, I need to leave a sample overnight at some point. 

I cut the zebra finch head into anterior (beak) and posterior (brain) region and mid-sagittally in both. so each tissue sample is about 0.5 X 1 X 1cm cube at maximum. 
 
I have also tried short time schedule compared the one i wrote in previous email, for similar sized  sample (zebra finch beak). However, it never worked. Thus i prolonged the each step for the latest sample.  
It would be great if you could provide me the proper time schedule.

rui 

> From: krns at regionsjaelland.dk
> To: ruio7 at hotmail.com
> Subject: SV: [Histonet] dehydration time for a relatively large sample
> Date: Wed, 20 May 2015 06:18:24 +0000
> 
> Hi Rui
> 
> Which processor unit do you use
> 
> to me it seems like a wrong protocol.
> 
> Maybe I can help you set up a better protocol - if you want - but then I need to know size og your tissue, processor unit and what kind of clearens you use.
> 
> Kind regards
> Karen
> Supervisor tissue processing
> Denmark
> 
> -----Oprindelig meddelelse-----
> Fra: Rui TAHARA [mailto:ruio7 at hotmail.com] 
> Sendt: Wednesday, May 20, 2015 7:11 AM
> Til: histonet at lists.utsouthwestern.edu
> Emne: [Histonet] dehydration time for a relatively large sample
> 
> Hi, 
> 
> I am wondering if prolong dehydration time with 95 and 100% ethanol would brittle the sample for paraffin sectioning. 
> 
> I have been trying to section adult zebra finch beak and processed several samples, however, 
> i failed to obtain a good section. It appears that the paraffin did not penetrated the tissue. This may be derived from incomplete dehydration and clearing before paraffin. 
> Because i have read somewhere that if the sample was sitting in 95 and 100% ethanol too long it would be brittle and be teared when sectioned.  I have processed only a beak (about 1 cmX0.5 mm). 
> Also when is an appropriate time to use vacuum? I am afraid that if i used it at 100% ethanol, the ethanol would evaporate....
> 
> so far I have tried; 
> fix overnight
> decalcified few days
> walk from water to 70% ethanol; overnight 
> 85%, 95% (2times change)  and 100 % (2 times change) ethanol; overnight   
> clearing; 2 days
> paraffin (2 times change); overnight
> 
> Any suggestion would be appreciated. 
> Thank you in advance, 
> 
> rui 
> 
> 
>  		 	   		  
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