[Histonet] How far will stain reach into a thick section on a slide?

[John Kiernan]

Sections of brain much thicker than 20um can be Nissl
stained or immunostained all the way through. For some
purposes 80-100um is the preferred thickness. Nuclei
(meaning groups of similar neurons) stand out clearly,
and individual immunostained axons or long dendrites
can often be traced over long distances, especially
if there are not too many, close together. 

Usually thick frozen sections are handled free-floating,
so that reagents can enter from both surfaces. If your
50um sections are stuck on slides, longer times may be
needed. For immunostaining it's usual to apply the
primary antibody for 24-48 hours at 4C and to include
a non-ionic detergent such as saponin or Triton-X to make 
holes in the cell membranes. Times in secondary antisera
and related reagents are only 30-60 minutes.

For Nissl staining use a weak solution of a cationic 
dye at pH between 3.5 and 4.0). Stain progressively and
wash in water buffered to the same or a higher pH. An 
acetate buffer is convenient and cheap. It can be very 
dilute but must not be even slightly more acidic than 
the dye solution. If you wash the sections in tap water,
do this is in the sink, with a rapid flow of water
through the slides. If the sections spend significant
time in an unbuffered solution of the cationic dye 
there will be some background (protein) staining
. 

A pure Nissl stain shows only DNA (cell nuclei) and RNA 

(in rough endoplasmic reticulum of neuronal cell bodies).
---
Historical interlude:
Franz Nissl (1860-1919) was a German "neuropsychiatrist"
who also did the histopathology of his patients. He
described normal and pathological neurons about 10 years
before the biochemical recognition of nucleic acids
and at least 50 years before cytoplasmic basophilia was
solidly attributed to to RNA in ribosomes. 
If you find this sort of historical stuff interesting,
click on
http://www.whonamedit.com/doctor.cfm/2465.html
for Nissl, the man and for links about his mentor,
poor old Johan Bernhard Aloys von Gudden (1824-1886),
who was probably murdered by an ungrateful patient,
King Ludwig II of Bavaria. The mad king and the 
neuropsychiatrist died together. von Gudden had been
sent by the parliament to certify Ludwig, who had
brought his country close to bankruptcy by building
fairytale castles and palaces. The buildings are 
still there, and tourism may have recovered the
losses incurred by Ludwig II.
---
Which dye?  Thionine, toluidine blue and azure A are 
good blue Nissl stains; neutral red is also good. 

Many people like cresyl violet, and there's a chemically 
related dye called Darrow red that was invented for Nissl 
staining (MM Powers et al. 1960 Stain Technol 30:83-88). 
I've never used Darrow red or seen it offered for sale, 
but its use appeared in publications in the 1990s. Darrow
red has also been used by biochemists as an enzyme
inhibitor. The dye is named for the late Mary Darrow, who
was the chief technician at the Biological Stain Commission's
laboratory in Rochester, NY in the 1930s, '40s and '50s.

Having found a suitable concentration and time for staining
your sections in your lab, rinse in the acetate buffer and
dehydrate in 3 changes of 100% alcohol. This will minimize 
loss of dye from the stained nucleic acids. Alcohol-water
mixtures extract most stains more quickly than 100% alcohol.

Several Nissl procedures exploit this, and use a graded
alcohol dehydration to differentiate overstained sections.
That's a waste of good alcohol. The specificity of staining
is determined largely by the pH of the dye solution. The
staining time will be shorter with a higher dye 
concentration, but for thick sections time is needed for
penetration. If the dye concentration is too high there
will be more unbound dye to wash out of the sections, and
this will end up in the dehydrating alcohols. Go for the
lowest dye concentration that fits into your working day.

John Kiernan
Anatomy, UWO
London, Canada
-----------------------------------------
CM Bush wrote:
> 
> Dear Histonet,
> 
> I am wondering what you can tell me.  We are staining thick sections of human brain.  The brain has been very well fixed in 10% NBF, and sections are mounted on slides.  We are using thick sections (50um) and Nissl or immuno staining.  Does any one know how deeply Nissl stain (or an IHC stain) can permeate tissue mounted on a slide?  Is, say, any tissue thickness greater than 20um (for example) moot as it will never be reached by the stain?
> Would staining floating sections be a more rational way to deal with thick sections?
> 
> Thank you vey much for the help.
> Clarissa
> 
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