[Histonet] dehydration time for a relatively large sample

Tue May 26 01:59:11 CDT 2015

Thank you for your advice!
I was going to try if the current sample that was embedded by a technician at the histological service center. This is the first time that i asked for an expert to prepare my sample. Then I sectioned the sample by myself. However, the muscles in the section always looks very dry or are unable to be penetrated with wax, thus the sections do not look that great.... I am not an expert at all for preparing histological sections, so if the sample embedded by an expert, I am not sure how to fix the problem. 
I may try it again following your time schedule, but i am a bit tight schedule to end this project. If i try i will let you know how it turns out. 

I currently reside  in montreal, Canada and am studying as a PhD student. 
I really appreciate your help. 

rui 

> From: krns at regionsjaelland.dk
> To: ruio7 at hotmail.com
> Subject: SV: [Histonet] dehydration time for a relatively large sample
> Date: Thu, 21 May 2015 13:24:56 +0000
> 
> Hi Rui
> 
> Sorry for a little late response. I tried to write to you yesterday but my internet failed.
> 
> Tissue is so much easier with a processor,  but life is not always easy and we have to do things the possible way ;-)
> 
> First I have to tell you, that I never have tried to process tissue by hand..... and I have never tried to process zebra finch.  BUT.... I have been helping lot of other people with protocols,  so I think I'm able to help you too. maybe we need to adjust the protocol. ... But lets see how it will work.
> 
> First.... make sure your tissue are well fixed before decalcination. 1 day in formalin. 
> Decalcinate the tissue as short time as possible but make sure it's with out calcium before processing. 
> 
> 70% ethanol 3 hours
> 96 % ethanol 5-7 hours
> 99 % over night ( about 12 hours )
> 99 % so it will fit your work ( change 99% 2 times)
> Clearens over night (about 12 hours )
> Clearens so it will fit your work ( change clearens 2 times )
> Paraffin 2-3 days ( change paraffin 3 times )
> 
> When you embed the tissue please notise if you see lot of small bobles. The bobles can be a sign of poorly infiltretet paraffin (water in the tissue)
> 
> Please let my know how things go, because we maybe need some adjustment. 
> 
> I would like to know where you are from. I have helped all over the would and it's fun to know.
> 
> Kind regards
> Karen
> it's___________________________
> Fra: Rui TAHARA [ruio7 at hotmail.com]
> Sendt: 20. maj 2015 15:44
> Til: histonet at lists.utsouthwestern.edu
> Emne: Re: [Histonet] dehydration time for a relatively large sample
> 
> 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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> > Histonet at lists.utsouthwestern.edu
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> 
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