Silverman's Modified Movat pentachrome

From:Jeff Silverman

Thanks to all those interested in the Movat pentachtrome modification.  The
reference is Histologic Vol II No 2- April 1972. Excellent for vascular
lesions and dissecting the stroma of any tissue and especially soft tissue
tumors. Poly Scientific R &D Corp in beautiful Bay Shore NY sells a Movat
kit but it is not quite the same as my method with regard to Nuclear and
elastica staining (I have no relationship)but to save time you should check
it out too and maybe make your own acid orcein Verhoeff. Feel free to
contact me for details or trouble shooting.

Jeff Silverman HT HTL QIHC (ASCP)
Pathologists' Assistant
Southside Hospital
Bay Shore NY
peptolab@hamptons.com

Microtomy: Paraffin sections at 4-6 micra.
Fixation:  Bouin's or 10% buffered formalin. If formalin is used, decerated
sections must be mordanted in Bouin's for 1H in a 56 degree oven and washed
well with running water to remove yellow picric deposits.

1.  Stain sections in 1% alcian blue dissolved in 1% aqueous acetic acid
for 20 min.
2.  Rinse in DH2O and place in 5% ammonium hydroxide in 95% ethanol for 10
min at 56C.
3.   Wash in running tap water for 2 min. Rinse in DH2O.
4.  Stain in acid orcein-Verhoeff working solution for 2 hours.
	Orcein-Verhoeff Stock Solutions
	Sol. A-- Orcein............................1.0 gram
		Ethyl alcohol 70%...........498 ml
		Hydrochloric acid, conc...1.0 ml
	Sol. B-- Hematoxylin....................8 grams
		Absolute ethanol.............160 ml
	Sol. C-- Ferric chloride..................9.6 grams
		DH2O..............................90 ml
	Sol. D-- Iodine.............................1.0 gram
		Potassium iodide..............2.0 grams
		DH20...............................97 ml.
	Working Solution:  Combine immediately before use in the following
order.
		Use once and discard.
		Sol A.....  25.0 ml
		Sol B...... 8.0 ml
		Sol C.....5.0 ml
		Sol D.....5.0 m
5.  Wash in running water for 3 minutes. Differentiate in a 1:4 dilution of
Solution C (2% ferric chloride) until collagen is clear and elastic stands
out. Wash again for 1 minute in tap water.

6.  Stain in woodstain scarlet-acid fuchsin mixture for 3 minutes.
	Woodstain scarlet-acid fuchsin mixture stock solutions:
	Sol A-- Woodstain scarlet NS conc (or Crocein scarlet MOO CI27290)
	  	................................0.1 gram
		DH2O...................... 99.5 ml
		Glacial acetic acid......0.5 ml
	Sol B--  Acid fuchsin..............0.1 gram
		DH2O..................... 99.5 ml
		Glacial acetic acid.... 0.5 ml
		Working solution:
		Sol A.........40.0 ml.
		Sol B......... 10.0 ml.
7.  Place in 0.5% aqueous acetic acid water for 30 seconds.
8.  Differentiate in 5% aqueous phosphotungstic acid for 3-10 minutes.
Control under the microscope until red color comes out of collagen and
stromal ground substance reappears as blue color.
9.  Rinse 0.5% acetic water for 30 seconds.
10.  Place in three changes of absolute ethanol, 1 min. each.
11.  Stain in 6% alcoholic saffron for 8 minutes.  Prepare the solution by
extracting 6.0 grams of spanish saffron CI No. 75100 in 94 ml. absolute
ethanol in an airtight bottle in 56-60 degree oven for 48 hours.  The
solution only works effectively if kept anhydrous with Drierite and can be
reused for
some time until staining intensity decreases.
12.  Dehydrate quickly in 2 changes absolute ethanol.
13.  Clear in xylene, three changes.
14. Coverslip with any resinous mounting medium.

Results:
Nuclei......................... dark purple to black
Cytoplasm....................pink to brownish to red
Elastic fibers................purple to black
Collagen and bone.........yellow to yellow green
Mucin, ground substance........varying shades of blue to blue green.
Epithelial mucins are blue and ground substance is blue-green owing to
collagenous staining by saffron admixed with the alcian blue-stained acid
mucopolysaccharide ground substance.
Muscle.................red with excellent delineation of longitudinal
myofibrils, cross striations
and intercalated discs.







<< Previous Message | Next Message >>