Re: [Histonet] Sihler's stain

From:Maria Mejia

Hello Dianne,

I have not only heard of this stain, I have done it on thick whole 
monkey extra
ocular muscle thick sections. The staining procedure is a simple one, 
don't be fooled - this is a challenging stain to do!!! The reference I 
have is titled
"The Sihler stain: a technique for studying peripheral nerves in whole-mount
specimens" by Bei-lian Wu & Ira Sanders. Over the course of several 
trials, I
have modified the methodolgy from the article to fit what I needed.

The following technical times are for whole thick monkey extra cellular 
**The total time required to stain varies & depends on the size & type 
of the tissue
sample - so testing should be done (before) doing on the real McCoy samples.
***Each step (exception is the running water step) - should use agitation!!!
Ideally, tissue should be perfused w/10% UNNEUTRALIZED formalin/DH20
prior to removing the tissue. Immersion fixation technique of fresh 
tissue should
also work. The slight amount of formic acid in formalin is less harmful 
in its
effects upon nerve tissue than a neutralized solution. Remember to 
change the
formalin fixing solution once a week - it takes 1 month or longer for 
larger whole-
mount tissue. However, for my thick tissue sample, I fixed for only a 
few days
to one (1) week (w/agitation).

1.  place tissue samples in tissue cassettes & in a container wash in 
gentle running
water - 30 minutes.
2.  remove samples from cassettes & place one tissue sample in each 
large clear
plastic well containing 3% potassium hydroxide w/0.2ml of hydrogen peroxide
in 100ml DH20. the clear plastic box w/lid has 15 to 25 wells -depends 
on the size
you buy.  each well is clearly ID.
**change this solution every other day & total time for tissue in this 
solution varies
from 4-6 days.
3.  specimen initially appears highly refractive, but will progressively 
lose this
property. check specimen under a dissecting microscope every day until 
the soft
tissue is faded, transparent & barely refractive while the  nerves & 
especially the
small nerve brances can be seen clearly as white fibers.
**KHOH affects nerve tissue to a certain extent, so this step should be 
as brief as
possible. the durration of this step varies w/each specimen size. may 
take 2 to 3
weeks for different wholemounts
4.  remove tissue from KHOH solution, place tissue again in cassettes & 
wash in
gentle H20 - 30 minutes.

Staining of Tissue: Use the Sihler II solution to stain the specimen.
5.  after washing, remove samples from cassettes & again place in 
plastic wells
that contain working Ehrlich's hematoxylin.
**this hematoxylin solution is very important!!! it must be at least 3 
weeks old and
NOT older that 6 months for this stain to work!!! I buy my Ehrlich's hem 
Electron Microscopy Sciences 215-646-1566. ask to speak w/the technical 
who makes the hematoxylin tell him what you need and to write the date 
on the bottle
when the hematoxylin was made, so you can keep track of its usage 
peak!!! the time
is important because it tends to under stain if not ripe enough & over 
stain if too old
& therefore more difficult to remove excess stain from tissue during the 
**solution should be changed every other day & agitate. total time for 
staining should
be about 4 to 5 days.
**check tissue every day under dissecting scope. could take 2 to 3 wks 
if staining
different thick wholemount tissue - don't know times for thinner sections.
The endpoint is when all nerves including the finest brances are stained 
dark violet
blue. for small or this specimens the amount of hematoxyling maybe 
decrease to as
little as 0.5 parts.

De-staining: use the Sihler I (working 0.5% acid solution)
6.  remove tissue from wells & place in tissue cassettes...again - & 
wash in gentle
running H20 - 10 minutes.
7.  after washing, return samples into wells w/Sihler I acid solution - 
check tissue
every 20 minutes & change solution if it turns blue!!! total time could 
take up to
2.5 hours - so check, check tissue under scope!!!
***check tissue by trans illuminating them while observing under the 
scope. end-point is when tissue has a transparent lavendar colored to 
clear soft
tissue w/dark violet nerves.
**if the nerve brances look faded or take on a yellow tint, the tissue 
is either over
de-stained or there is too much acetic aicd in the Sihler sol. if this 
occurs, tissue
must be washed thoroughly w/tap H20 & re-stained in Sihler solution II & 
repeat the de-stain step again - this time use less glacial acetic acid.
**but I found that my modified method was suitable for smaller, thinner 
or lightly
stained tissues samples.


8.  after de-staining the tissue is acidic & needs to be neutralized. 
place sample in
tissue cassettes...again, wash tissue thoroughly in running - 1 to 1.5 
9.  remove sample from cassetts & return to  wells with freshly made 0.05%
lithium carbonate (agitate).
**check every 15 minutes & change solution  if it turn pinkish color. 
leave samples
in this solution until the black-blue fibers colored nerves turn to 
violet-blue -
about 1.5 hours


10. remove samples from wells & place in cassettes again to wash in 
running H20 -
for 30 minutes to 1 hr. (I did 30 minutes).
11. remove samples from cassettes and place in several wells that 
contain increasingly
graded glycerin (buy a good brand grade of glycerin) - start with:
 >40% glycerin/DH20 - overnight.
 >60% glycerin/DH20 - overnight.
 >80% glycerin/DH20 - 1 to 2 nights
 >99% glycerin - 2 to 3 nights
and finally 100% glycerin - 4 nights.
** can store tissue in this last step, just add a few grains of thymol. 
change glycerin
every 4 to 6 months using fresh 100% glycerin w/thymol.

WOW, that's it!!!

Maria Bartola Mejia
Smith-Kettlewell Eye Research Institute
San Francisco, CA 94115
Email: maria

Dianne Holmes wrote:

>I have been asked to use the Sihler technique to stain nerves in the trachea.  I have only 'heard' of this method and would like some input from someone who has actually used  it.  The reprint I was given to refer to is a 2001 experiment on rabbit eyes and it took up to 3 months to process the tissue !!  Any help would be appreciated.
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