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.
http://students.washington.edu/ckthomps/image1.jpg
This image illustrates the blotchy staining I'm seeing. At higher power,
labeled cells in lightly stained areas are visible but very faint.
http://students.washington.edu/ckthomps/image2.jpg
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.
Thanks,
Chris Thompson
