[Histonet] mounting and staining celloidin-embedded whole brain sections

[Rittman, Barry R]

Jim
You are now talking about real old fashioned histology!

I have cut, mounted and stained Low Viscosity Nitrocellulose sections
(of human embryos) up to 130 microns thick.
I am assuming that you are talking about LVN rather than celloidin but
the same general principles apply.

Stain, using very dilute solutions, differentiating agents etc.
Can dilute Ehrlich's hematoxylin to 1 -5 % using glass distilled water.
Then need to filter. Solution only keeps a day or so. Stain for up to
several hours. Differentiation required is usually minimal. Same
principal for eosins etc.
This is best done using free floating sections.
Sections that are stuck to slides before staining tend to have areas
that lift from the slide and give patchy staining in those areas.
These can be processed as usual through alcohols up to 70%.
90% ethanol and above will soften and then dissolve LVN and celloidin.
To prevent this go from 70% to a mixture of absolute ethanol and
chloroform.  Can use a variety of mixtures but generally need at least
15% of the mixture to be chloroform. Carful at this step not to have
excess humidity in the room or breathe too heavily on the sections.
If you use xylem or toluene then sections become brittle and curl.
I have used terpineol also known as oil of lilacin.
This will allow sections to remain supple and sections can be left in
this some several minutes.
This can be done with free floating section or can place the section on
slid and add the terpineol directly.
Terpineol is thick and so will not flow off slides and you will see a
lot of convection currently. Would recommend leaving 5-10 minutes at
least.
Carefully drain off terpineol and then remove excess terpineol remaining
on the slide using bibulous paper (or lens tissue) with several layers
of  filter paper or paper towels to absorb terpineol. 
Terpineol is miscible with most permanent mountants.
The major problem is keeping sections flat. I have used an excess of
mountant and after applying coverglass have weighted this down with
brass weights.
I had some made, if you need any please let me know as I have a half
dozen or so that I could send you.
Don't be cheapskate with the mountant or you will have bubbles creeping
in from the side.
One small point, if you need to use any enzymes on the sections then you
will have to remove the celloidin as most proteins cannot penetrate the
pores in the celloidin. Also some stains such as Celestine blue, luxol
fast blue etc. will permanently stain the celloidin and also render it
insoluble.
Hope this helps.
If you need more details please contact me.
Barry


Barry Rittman, PhD
713- 500 -4134  Office


-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of jstaruk
Sent: Thursday, March 27, 2008 10:06 AM
To: 'Histonet'
Subject: [Histonet] mounting and staining celloidin-embedded whole
brainsections

All,

 

I have 160 sections of celloidin-embedded Human brain sections cut at
50um
which now have to be mounted on glass and stained.  Is there anyone out
there who's done this before?  My main concern is how to mount the
sections
to the glass and also staining methods (before or after mounting?).

 

Thank you

 

PS, for all of those who requested an AFB control block from me last
week,
they were mailed out yesterday.

 

Jim

 

_____________________

     Jim Staruk

Mass Histology Service

www.masshistology.com


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