[Histonet] RE: Uneven Staining

[Debbie Faichney]

Liz,

Could it be due to the presence of residual wash water or previous solution diluting the stain to give patchiness?  On the same thread, insufficient agitation of the slides in the solutions?  You do not say if staining is by hand or automated as this may be a contributor.
Whilst I have no experience of the staining method in use, I stain by hand and therefore able to ensure the previous solution is well drained prior to placing in the next.  The slides are also dipped in and out( once or twice) to ensure the previous solution is washed off as well as allowing the stain to be evenly distributed  amongst the slides.

I hope you are able to resolve this frustrating issue.

Kind regards

Debbie 

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Elizabeth Cameron
Sent: 27 November 2012 19:13
To: Rittman, Barry R; histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] RE: Uneven Staining

Barry,
Thanks for the suggestion.  We are generally only able to get about 10-15 sections in a ribbon due to the nature of the tissue, and the pale staining last for 30+ sections at times.  We are also cutting on multiple microtomes (one microtome/kidney, but MANY kidneys!), so all microtomes would have to be affected.
No ideas on this one, so I appreciate any far-reaching thoughts!
Thanks,
Liz

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Rittman, Barry R
Sent: Tuesday, November 27, 2012 1:46 PM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] RE: Uneven Staining

Elizabeth
as the sections are evenly stained but vary in their staining from slide to slide, one suggestion is that section thickness varies. I know that this is reaching but if you section a ribbon and then place this on the water bath and then cut the next portion of ribbon then perhaps the microtome has a problem with the pause between cutting.
If this is the case try cutting a long ribbon and then mounting the separate slides.
Again, know this is reaching but difficult to see what it could be.
Barry


From: histonet-bounces <@t> lists.utsouthwestern.edu [histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Elizabeth Cameron [Elizabeth.Cameron <@t> jax.org]
Sent: Tuesday, November 27, 2012 12:35 PM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] Uneven Staining

About a year ago, I posted about issues with staining mouse kidney sections (see below for original post).  We realized that part of the problem was the sections drying during staining, which has been resolved, but we are still having some issues with some slides staining well and others being very pale.  This time it seems to be consistent across the slide, so I know it is not a drying issue.  They are serial sections, so they are all cut together and stained together, and they are from the same animal.  I don't believe deparaffinization is an issue - we have tried longer times with fresh solutions and it doesn't make a difference.  We have also tried re-staining the slides after letting them sit in xylene overnight, and there is no difference in the staining.  I am now thinking that it may be related to how long the sections soak or how long they sit on the waterbath.  They are NBF fixed, and there is a fine line between soaking well for hydration and oversoaking, resulting in swelling.  Has anyone experienced anything like this?  Any other ideas?  I am at a loss, and I'd really like for this to work!
Thanks,
Liz

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!
>



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