[Histonet] Histonet

[Hugh Luk]

Sophia,

Holes like you mentioned, are probably from the polymers or impurities in your embedding wax separating.  Gayle Callus mentioned this several times (see histosearch), and recommended "Stirring" the embedding paraffin every so often (before every use).  It could also be your tissue processing.  Check to make sure it is properly processed.  Embed without letting the wax solidify before doing so.  You may have a bad batch of reagents?

As for the stainer, Hazy nuclei, as long as you are sure your hemotoxylin is fine, are indicative of incomplete deparaffinzation, which could mean your OVEN or your reagents.  It could also be your acid/base solutions.

Note:  Have you started a new lot # of wax?  How about xylene (xylene-substiture) or alcohols?  I have heard people saying X-brand paraffin was having problems with impurities that could cause these problems.  I also heard it was solved last year. 

Also, your stainer has very little leeway in deparaffinzation.  You might have to do things by hand until this problem is resolved.  You know, deparaffinize to water using the longer method, then finish the H&E in the stainer.  Does this help?


Good luck (whoa, you have your work cut out for you),
Hugh
UH Cancer Center
Hawaii


> Message: 6
> Date: Wed, 27 Mar 2013 06:15:13 -0700
> From: Sophia Lin <lins0701 <@t> gmail.com>
> Subject: [Histonet] Wholes in tissue 
> To: Histonet <histonet <@t> lists.utsouthwestern.edu>
> Message-ID: <B099F758-6E27-4C58-AE00-6FE65EF1D234 <@t> gmail.com>
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> 
> We noticed that once a section was placed on the water, the section has wholes that appear. They are not present when sectioning (not seen by the naked eye) but appear when the tissue is stretched. Is this an embedding issue? The "wholes" appear next to the tissue. This hasn't happened before just recently. 
> 
> Any thoughts?
> 
> Sophia 
> 

> 
> ------------------------------
> 
> Message: 17
> Date: Wed, 27 Mar 2013 09:26:45 -0700
> From: Sophia Lin <lins0701 <@t> gmail.com>
> Subject: [Histonet] linear stainer hematoxylin staining issues
> To: histonet <@t> lists.utsouthwestern.edu
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> 
> We have the Hacker linear stainer, and for the past two weeks we had clean,
> crisp H&E staining. However, last Friday, something happened and our H&E
> stains no longer look crisp, but instead have a hazy, unreadable look.
> Nothing different has been done, and we have troubleshot for every possible
> scenario. After a clean set up, we noticed that the hematoxylin is not
> staining very well. We have it set for 2 minutes with agitation for a
> uniform staining. Hematoxylin is Harris Special (mercury free) from MCC and
> the eosin is with phloxine b from AMT. After the hematoxylin (2 buckets for
> total of 4.5 minutes), we use 1.5% acetic acid. It seems that the
> hematoxylin is not staining well.
> 
> Any tips? We may need to consider increasing a bucket for hematoxylin?
> 
> Thanks,
> 
> Sophia
> 
> 
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> End of Histonet Digest, Vol 112, Issue 30
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