[Neuroscience] blotchy Nissl staining

Chris Thompson via neur-sci%40net.bio.net (by ckthomps from u.washington.edu)
Fri Mar 16 16:28:22 EST 2007

I'm new to this list, so please bear with me. I'm having some big 
problems with some Nissl staining. I have two avian brains, perfused 
with heprinized-saline, fixed with 4% paraformaldehyde, and postfixed 
for 24hr in PFA. I embedded the brain in gelatin, fixed the gelatin in 
10% NBF, and sectioned the brains on a sliding microtome at 40um. I put 
the sections into saline solution for a few hours, mounted them onto 
gelatin-subbed slides, put them on a slide warmer for a few hours, and 
let them air dry overnight.

Protocol for Nissl stain: 5min H2O  -> 5 min thionin -> dip H2O -> dip 
70% EtOH + acetic acid -> 10-12 sec 70% EtOH -> 2X 1.5 min 95% EtOH -> 
3X 2 min 100% EtOH -> 3X 3 min xylene -> coverslip with DPX

Both brains show blotchy staining for Nissl, but one is much worse than 
the other. There is some consistency across sections, which might 
suggest the problem happened before sectioning, but some sections 
adjacent to bad sections are fine. I've uploaded images of two adjacent 
sections to my account to illustrate what I'm talking about.


This image illustrates the blotchy staining I'm seeing. At higher power, 
labeled cells in lightly stained areas are visible but very faint.


This an image of an adjacent section that shows typical staining. The 
light C-shaped arc that runs through the image is an axonal tract that 
is typical and is not the blotchy staining I'm seeing.

I'm sort of at a loss to explain this. I've stained dozens of brains 
with this protocol and I've never seen this phenomenon. Any advice would 
be appreciated.


Chris Thompson

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