[Histonet] Uneven Staining

[Geoff McAuliffe]

On 12/14/2011 9:55 AM, Elizabeth Cameron wrote:
> We are doing a Hale's colloidal iron stain (no counterstain) on serial sections of mouse kidneys.  We are staining an entire kidney at a time (about 90-150 slides), and after many successful runs, we are now finding some slides in each batch with very uneven staining.  Half of a section will stain as it should, and the rest of the section is very pale.  It seems to be in a similar area from one section to the next, but not exactly the same area.  It does not look like a deparaffinization issue.  There may be two or three slides in a row like this, or just one section on a slide, followed by 30-40 that look fine.  The sections are 6 microns. Any ideas on why this might be happening?  Thanks!
>
> Elizabeth M. Cameron, HT, QIHC (ASCP)
> The Jackson Laboratory
> Bar Harbor, Maine
> (207) 288-6561
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>
Hi Elizabeth:

It seems like every time someone posts an "uneven staining" question, 
the solution (not just the answer) is always incomplete deparafinization.
Try fresh xylene, minimum 2 changes 5 minutes each, and fewer slides in 
the rack and some agitation (of the slides, not of you).

If that does not solve the problem incomplete fixation might be the 
culprit.Perfused tissue? Fixed how long in ?

Geoff

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Geoff McAuliffe, Ph.D.
Neuroscience and Cell Biology
Robert Wood Johnson Medical School
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