RE: Smooth Muscle, Collagen and Elastin Staining at once

From:"Monson, Frederick C."

Morning Rafael,
	You may get some different advice, but I will simply state that I
worked in Urology at the University of Pennsylvania for 12 years doing
histology.  My advice is the following.

	Since you will NOT have interference from mucous-secretions in the
cavernous tissue, I recommend the use of Gomori's Aldehyde Fuchsin (Ref:
Lillie, R.D.(1965), Histopathologic Technic and Practical Histochemistry,
McGraw Hill, NY, NY, p.556-557.

		1 each		250ml serum bottle with Teflon-lined screw
		1.0g 		Basic Fuchsin (C.I. 42510)
		200ml		70% ethanol
		2ml		HCl (Concentrated) [CORROSIVE &
		24-48hr at room temp.	I always make on Friday for use
starting Monday.
		Store at 4 degrees - useful for 3-6months depending on
frequency of use and efficacy proved by testing bi-weekly.

		1.	Bring sections to 70% ethanol
		2.	Immerse in Aldehyde Fuchsin* at room temperature for
up to 30min
		3.	rinse in multiple changes of 70% ethanol to remove
excess dye
		4.	bring sections to water
		5a.	dye with H&E using routine method with
differentiation and stabilization of the hematoxylin (I use Ehrlich's acid
hematoxylin and alcoholic Eosin!)
		5b.	you may dye with Mallory's Triple Stain (Part II)**
for at least 30min 		6.	differentiate the Mallory's Part II
with several 90% ethanol [to prevent formation of precipitate(?)] until the
collagen is the blue you like.
		7.	follow with Eosin for the smooth muscle (easier than
the Acid Fuchsin which tends to wash out easily) 
		8.	dehydrate and mount (I use Damar/Xylene)

*	Not all batches of Basic Fuchsin or Pararosaniline work in this
method.  I use an old lot that was manufactured something like 25yr ago or
more.  I have several bottles that I have kept, because it is the best I
have found for the Gomori Aldehyde Fuchsin method.(Harleco, Fuchsin - Basic,
Lot 8242, (C.I. 42510), BSC Certification No. LF-56 [older than I

**	Mallory's Part II
	100ml		1% aqueous Phosphomolybdic acid
	0.5g		Aniline Blue (water soluble)
	2.0g		Orange G

	Results:	nuclei orange, collagen fibers blue, elastin fibers
magenta, smooth muscle red/orange.  

	        The advantage of this method is the deep magenta color of
elastin which can be readily seen against the blue of collagen, the red of
cytoplasm(muscle) and the orange of nuclei.  

	         I have sent two images to the Histonet.  One is a 10X image
of an elastic lamina that has been buried in the wall of a urinary bladder
14D following outlet obstruction.  The dyes are Gomori's Aldehyde Fuchsin
followed by H&E.  A 40X image is also included of isolated elastic fibers
from the same section.  I think that these examples demonstrate the quality
of this approach.  Fixation was in 4% formaldehyde in PBS at around pH 7.4.
Please note that the obstruction causes an acute overdistension of the
bladder (vol from 20ml to as much as 100ml volume).  A scar develops within
the peritoneum which, in the rabbit, unlike the human, covers almost the
whole bladder.  Within weeks, the scar becomes frequented by myofibroblasts
and subsequently bundles of smooth muscle appear.  See Sternberg, Histology
for Pathologists (eds 1 or 2) for a chapter on this genre of cell.

	To reference the concept of an elastic lamina I must refer you to:
"Use of a Previously Undescribed Elastic Lamina of the Serosa to
Characterize Connective Tissue Hypertrophy of the Rabbit Bladder Wall
Following Partial Outlet Obstruction",  Monson, F. C., Goldschmidt, M.H.,
Zderic, S.A., Ruggieri, M.R., Levin, R.M. and Wein, A.J., Neurourol and
Urodynam, 7:  385-396; (1988).  It turns out that one sees elastic fibers in
definitive lamina almost everywhere using the Gomori AF method.

	Regards, and you may ask questions,

	Fred Monson

Frederick C. Monson, PhD   
Center for Advanced Scientific Imaging
Schmucker II Science Center
West Chester University
South Church Street 
West Chester, Pennsylvania, USA, 19383
Phone:  610-738-0437
FAX:  610-738-0437
CASI URL:  http://darwin/
Visitors URL:

> ----------
> From: 	Rafael Badalyan
> Sent: 	Wednesday, April 3, 2002 11:40 AM
> To:
> Subject: 	Smooth Muscle, Collagen and Elastin  Staining at once
> Dear Colleagues
>     I am Rafael Badalyan, MD urologist in postgrad. research from Armenia.
> In my current work I can across with penile erectile tissue evaluation. 
>     Could you please help me with an advice? I have collected cavernous
> penile tissues, fixed them with formaline 10% and imbedded in paraffin.
> Now I do not know how, with which method to stain them? What I am going to
> evaluate is the correlation of smooth muscle, collagen and elastic fibers.
> Which staining method could you advice me for that? What I have found from
> different sources is the modified elastic trichrome stain. But this method
> seems not so easy for our laboratories, and the main problem is the
> Biebrich Scarlet, which is impossible to find in Armenia. If you find this
> method a useful for current situation, could you please say which stain I
> can use instead of Scarlet? Can it be changed to acid fucsin? Or if you
> can sudjest any other more useful and appropriate technique, I would be
> very much thankful to know. 
>  2) And as I have a chance to consult with you all, I also would like to
> ask you the second question: whether it is possible to perform Western
> Blot analysis for , TGF-B1, Bcl-2, p53, HIF, with evaluating cavernous
> smooth muscles, which are taken from cadavers, or it should be only
> biopsies from patients????
>     Thanks a lot for finding time to review this mail. I hope and wish to
> receive your advice.
>     With best regards
>     Sincerely,
>     Rafael Badalyan, MD
>     NIH, 375051 Yerevan, Armenia
>         Komitas av. 49/4
>     Tel: 3741 534163
>     Mail:

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