[Histonet] Bouin's, formic acid & and sirius red staining

Wed Apr 15 19:29:33 CDT 2015

Hi Tryrone
A Mass Trichrome stain will stain up the collagen in the diseased kidney. If the tissue has already been fixed in Bouin's Solution you can omit the picric acid step. Another good stain is the Curtis's Van Gieson Stain. Ive sent you the protocol for botht he stains. Hope it helps.   

Masson Trichrome

Principal
This method depends on the special action of phosphomolybdic acid when combined with red aniline dye. The acid lifts the colour first from the collagen and only later from the cytoplasm. The differentiation is stopped at the point where the Collagen only is colourless, and then another aniline dye (light green) is added to stain the collagen

Reagents:	

1.	Ponceau Acid Fuchsin 
Warning: Suspected Carcinogen – see MSDS
Acid fuchsin (Sigma Aldrich CI 42685)     0.1g
Ponceau de xylidene (CI 16150) 	  	1g	                        
Distilled Water                       		99ml
When dissolved, add 1ml Glacial Acetic Acid

2.	Light Green
Warning: Suspected Carcinogen – see MSDS
Light green 	(CI 42095)			    2g	
Glacial Acetic acid 				  2ml
Distilled water 				98ml

3.	Weigert's Iron Haematoxylin or Celestine Blue/Haematoxylin

4.	1% Phosphomolybdic Acid
Phosphomolybdic acid			5g
Distilled water 				500ml

Procedure:	

1.	Bring sections to distilled water.   	
2.	Celestine blue 					10 minute.
3.	Wash well in water.
4.	Harris' haematoxylin 				10 minutes.
5.	Differentiate and blue                	
6.	Ponceau acid fuchsin solution 		10min
7.	Quick rinse in tap water
8.	1% Phosphomolybdic acid  			5min	
9.	Do not rinse in water
10.	Light green solution 			2min 
11.	Rinse excess stain rapidly in water.
12.	Dehydrate, clear & mount

Results:          
Collagen, mucin                                	green
Muscle, Fibrin                                            	red
Nuclei                                                           	purple

Curtis's Van Gieson Stain

Principle:
This solution is based on the same formula as Van Gieson with the exception of the use of Ponceau S rather than acid fuschin. Glacial Acetic Acid is used rather than hydrochloric acid to sharpen the staining results.

Reagents: 

1.	1% aqueous Ponceau S 
Ponceau S (CI 27195)				0.5g
Distilled water					50ml

2.	Curtis’s Van Gieson
1% aqueous Ponceau S 			10ml
Saturated aqueous picric acid      		90ml
Glacial Acetic Acid 	                         		1.5ml

3.   Celestine Blue – Haematoxylin procedure

Procedure:
1.	Dewax and hydrate sections.
2.	Celestine blue 5 minute wash.
3.	Wash well in water.
4.	Harris' haematoxylin 5 minutes.
5.	Wash in water and blue (do not differentiate).
6.	Curtis Van Gieson stain 10 minutes (do not wash).
7.	Rinse in absolute alcohol 
8.	Dehydrate, clear and mount.

Results: 
Collagen                 		Red
Muscle, other tissues    		Yellow
Nuclei                   		Blue

Linda Prasad | Senior Scientist | Histopathology
t: (02) 9845 3306 | f: (02) 9845 3318 | e: linda.prasad <@t> health.nsw.gov.au | w: www.schn.health.nsw.gov.au

Cnr Hawkesbury Road and Hainsworth Street, Westmead, NSW Australia
Locked Bag 4001, Westmead 2145, NSW Australia

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-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Tyrone Genade
Sent: Thursday, 16 April 2015 7:10 AM
To: histonet
Subject: [Histonet] Bouin's, formic acid & and sirius red staining

Hello,

Some questions regarding Bouin's solution.

I was told, back when I was doing my PhD and new very little, that I should fix my fish in Bouin's as it will decalsify the bones. Well, Bouin's fixed fish were easier to cut than PFA fixed fish... but I read today that by adding formic acid the decalsification is better. (I must confess, that after Buoin's fixation I still had to soak the tissue face in some dilute nitric acid now and then...) Another reference said that the formaldehyde should be replaced with formic acid. So which is it: add formic acid or replace formaldehyde? And if the former, how much do you add?

Second question: a colleague and I want to stain for collagen in diseased kidneys. The fixative of choice for soft tissue is Bouin's... But the staining protocol called for a solution of picric acid and sirius red. Is the picric acid needed if I haven't washed the picric acid from the Bouin's fixation out of the tissue? I was told once that the picric acid was for contrast... Is this BS? Does the picric acid play an important chemical role in the staining? I would like to avoid the need for a bottle of saturated picric acid on the lab shelf here in Iowa where the winter low humidity desiccates everything... I'm hoping this protocol, http://www.ihcworld.com/_protocols/special_stains/sirius_red.htm , can be modified to omit the picric acid.

Thanks
--
Tyrone Genade
Orange City, Iowa
tel: (+1) 712 230 4101
http://tgenade.freeshell.org
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