[Histonet] Nuclear Bubbling

Rene J Buesa rjbuesa at yahoo.com
Thu Feb 18 09:31:16 CST 2016

Essentially incomplete deparaffination shows a different pattern and is not limited to nuclei only.Again, this is what the consensus is:1- there is ALWAYS water left underneath the section2- that water HAS to be eliminated before the section is dried in the oven3- the best sectioning practices call for placing the slides (with the just fished sections) in a vertical position resting on their shortest end (1 inch) for a few minutes after the section is fished from the water bath. At my lab the techs were required to do that and their used to rest the sections around the water bath.4- AFTER the sections have been completely drained then they were either to the oven (60ºC for at least 7 min. or to the autostainer, we used a Sakura autostainer) and the sections were stained.
Which is the consensus as to what happens: when there is water underneath the whole section and it goes into the oven without properly drainage the HOT water will dissolve the nuclear contents something that it will NOT happen with the other tissue components (such as the connective tissue or nerves) because their chemical components are insoluble). The nuclei contain soluble chemical components and, as such, they will be dissolved in the hot water and if they are dissolved, they cannot be stained and will determine an image similar to an empty bubble, hence the "nuclear bubbling" artifact.
I have proven this mechanism by staining sections with different degrees of drainage (measured in minutes after the sections were fished) and the results were consistent with the explanation I offered above. The "ideal" drainage time is 7 minutes. Just try it yourself to dispel any of your doubts.

    On Thursday, February 18, 2016 3:00 AM, "Hoekert, Willem via Histonet" <histonet at lists.utsouthwestern.edu> wrote:

 Could it be due to incomplete deparaffinization? 


Van: Vickroy, James via Histonet [histonet at lists.utsouthwestern.edu]
Verzonden: dinsdag 16 februari 2016 18:10
Aan: histonet at lists.utsouthwestern.edu
Onderwerp: [Histonet] Nuclear Bubbling

Struggling to find an answer.  We do a lot of GI biopsies in our lab.  Sometimes they look wonderful without any nuclear bubbling, other times the bubbling is pretty intense.  Since nuclear bubbling is often attributed to incomplete fixation we of course have investigated the fixation times.  I do not find that the problem is fixation.  In fact some of the biopsies end up fixing for 48 hrs before processing. (weekend).  There was a suggestion last week or so that there might be water trapped under the slides after cutting and before staining.  I really thought that this might be the issue however I'm not sure at this point.  Extra drying seems to help but sometimes slides side by side are so variable, one with bubbles and one without.  I also don't believe the problem is in the processing schedule since the problem has shown up on both a rapid and a normal schedule. (therefore longer dehydration, clearing, etc.)

I am wondering if anyone else has worked with this issue.  Here are my questions:

1.        Could it be something that is happening with the tissue before it gets to the lab?  Usually a delay if fixation  causes other artifacts but not bubbling.  Could it be heat from the GI procedure?

2.      We do use blue sponges for our biopsies.  I know some say get rid of the sponges but has anyone seen this problem caused by usage of sponges?

3.      What about the heat stage in our Prisma stainer?

I am really getting frustrated.  Pathologists never complain however I would rather all of the tissue did not have the "nuclear bubbling".  Again we only do biopsies so I really don't think the standard old " not enough time in formalin" is the issue.  I have even wondered about variables such as we use recycled formalin, recycled Clearite III.

Any suggestions?


Jim Vickroy
Histology Manager
Springfield Clinic, Main Campus, East Building
1025 South 6th Street
Springfield, Illinois  62703
Office:  217-528-7541, Ext. 15121
Email:  jvickroy at SpringfieldClinic.com<mailto:jvickroy at SpringfieldClinic.com>

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