Hi Kathie,

Sounds like the problem could be in your differenciation step.  When I used
to do the stain I did not leave the slides in the ferric chloride for any
set of time. I would dip the slides, one at a time, until I got a grey
backround on the tissue.  Leaving the tissue in the ferric chloride too long
could take the stain out.  Also you need to make the Verhoff's working
solution fresh each time. Using the solution over and over will not give you
good staining.

Nancy Weber
Research Associate
Veterinary Clinical Sciences
Ohio State University
----- Original Message -----
From: "Kathie Berghorn" 
To: "HistoNet Server" 
Sent: Tuesday, October 16, 2001 6:15 PM
Subject: Help needed with Elastin staining


Histonetters,
A couple months ago I wrote asking how to do the Elastin
stain.  I received instructions (thank you very much!) and now have
experience with it .  BUT I am having a big problem with it -- lack
of consistency.  Slides cut at the same time, from the same animal,
same tissue (lung) have been used to work out the conditions in our
lab.  One time it will work beautifully, the next time using the same
times -- there is NO staining.  None. I just don't get it.  I have
included the procedure and solutions below.
Can any one give me some insight as to why this is happening?
FYI... lung tissue was immersion fixed in 4% paraformaldehyde prior
to paraffin embedding which was done by our histo lab.  I thank you
in advance for your help.

Kathie


Verhoeff Stain to visualize Elastin

. Rinse in Distilled Water. Stain in Verhoeff's working solution for 30
minutes.
. Rinse in tap water until clear.
. Differentiate if 2% Ferric Chloride from 2-8 minutes (monitor it under
scope) - we used 5 min
. Rinse in Tap water until clear.
. Then dehydrate, clear & mount.

Solutions for the Verhoeff portion

Verhoeff Stock       20g Hematoxylin (CI-75290) / 400 mL 100% ETOH
(filter after dissolved)

10% Ferric Chloride  26g Ferric Chloride / 260 mL DH20

Verhoeff Working Stain 330 mL Verhoeff Stock / 130 mL 10% Ferric chloride /
130 mL Lugols Iodine

2% Ferric Chloride 120 mL 10% Ferric Chloride / 480 mL DH20