Attwood's Stain

From:Rainbow Ron

Hi Christi,

Here is our method. 

Good luck

Regards

Ron Rainbow
Scientist in Charge
Department of Anatomical Pathology
Royal Hobart Hospital
GPO Box 1061L
Hobart
Tasmania
Australia
Ph 0362228771
Fax 0362228191
Email:  ron.rainbow@dchs.tas.gov.au



Department of Anatomical Pathology
Royal Hobart Hospital

ATTWOOD'S ALCIAN GREEN - PHLOXINE TARTRAZINE METHOD FOR EPITHELIAL SQUAMES
AND MUCONIUM IN FOETAL LUNG

SAFETY PRECAUTIONS:
	CAUTION: This department's policy is to treat all dyes as potential
carcinogens.
	Use a fumehood and protective clothing when handling hazardous
chemicals.
	A laboratory gown, gloves, safety glasses and dust mask is a minimum
standard.

PRINCIPLE:
	Any muconium or cartilage  that is present is initially stained with
alcian green. All other tissues
 	are then stained red with phloxine. Phloxine is progressively
differentiated by tartrazine in
 	cellosolve, first from muscle followed by connective tissue and
finally from red blood cells. The
 	epithelial squames as well as fibrin retain the phloxine stain.
SECTION:
	3mm paraffin section.
CONTROL SECTION:
	Foetal lung with epithelial squames present.
REAGENTS:
	1.  1% Alcian green (2GX) in 2% acetic acid.
	2.  0.5% Potassium permanganate:  Prepare fresh before use.
			CAUTION:	Strong oxidising agent. Toxic by
inhalation or ingestion.
					Corrosive. Avoid contact and
inhalation.
	3.  0.5% Oxalic acid.
			CAUTION:	Severe irritant and corrosive. Very
toxic.
					Avoid contact and inhalation.
	4.  Mayer's haematoxylin - Refer to method H1 in this manual for
reagent preparation.
	2.  0.5% Phloxine in 0.5% aqueous calcium chloride- Stable for 3
wks. Filter before use.
			CAUTION:	Phloxine is highly toxic.
	3.  Saturated tartrazine in cellosolve (2-ethoxyethanol).
			CAUTION:	Cellosolve is a teratogen. It is
flammable, toxic and
					an irritant. Avoid all contact.
		Saturation point of tartrazine in cellosolve is approx.
0.3%.

METHOD:
	1.  Dewax sections in xylene and hydrate through graded alcohols to
water.
	2.  Stain in alcian green solution --- 2 min.
	3.  Rinse in distilled water.
	4.  Oxidize in 0.5% potassium permanganate --- 2 min.
	5.  Decolourize in 0.5% oxalic acid --- 2 min.
	6.  Stain in Mayer's haematoxylin --- 5 min.
	7.  Wash in running tap water, 'blue' in ammmoniated water, wash in
running tap water.
	8.  Stain in phloxine solution using a coplin jar --- 20 min.
	9.  Rinse in distilled water, then in cellosolve (Refer to Safety
Cautions).
          10.  Differentiate in sat. tartrazine in cellosolve, checking
microscopically until phloxine is
	     removed from the red blood cells. Rinsing in water prior to
checking is recommended.
	     Continue with steps 9 & 10 until differentiation is complete
--- usually 15-20 min.
          11.  Dehydrate in cellosolve, clear in xylene and mount using the
automated Tissue-Tek 
	     Coverslipper or with a resinous mountant.



Issue Date: 04/10/1999	Amendment Date:	Review Date:19/7/01, 2/5/02
Authorised By: RD Rainbow/ T Van Galen	Amended By:	Reviewed By: RD
Rainbow
Routine Staining Method Manual
Page 1 of 2
Department of Anatomical Pathology
Royal Hobart Hospital

ATTWOOD'S ALCIAN GREEN - PHLOXINE TARTRAZINE METHOD 
FOR EPITHELIAL SQUAMES AND MUCONIUM IN FOETAL LUNG
(CONTINUED)

RESULTS:
	Nuclei --- blue / black.
	Muconium, mucin ground substance of cartilage, mast cell granules
--- green.
	Red blood cells, collagen --- yellow.

NOTE:
	Polyphenyl yellow 8G or lissamine flavine in place of tartrazine has
been used to facilitate
	differentiation. The phloxine staining of the squames has been
retained after overnight immersion
	in either solution despite phloxine removal from collagen, cytoplasm
and red blood cells in 1 hour.

REFERENCES:
	1.  Luna L.G. (1992) Histopathological methods and color atlas of
special Stains and Tissue
	     Artifacts; American Histolabs,Inc.; pp.242.
	2.  Attwood H.D.:The Histological Diagnosis of Amniotic Fluid
Embolism. Journal of Pathology
	     and Bacteriology. Vol.76; pg211; 1958. (Refer to laboratory
journal copy, section F).






























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