[Histonet] Verhoeff/ Trichome procedure

[Fail, Mildred M.]

ACTION  DATE    INITIALS








MODIFIED MASSON’S




PURPOSE:
To identify elastic in addition to connective tissue (collagen and muscle).

PRINCIPLES:
Masson's Modified is the combination of Masson's Trichrome and Verhoeff's Van Gieson (VVG) stains.  The stain procedure is the same as Masson's Trichrome with the addition of a VVG procedure to stain elastic fibers.  After the elastic fibers are stained, we continue on with the routine Masson's Trichrome stain.

FIXATIVES:
        10% neutral buffered formalin, B-3, B-5, Zenker’s, or Bouin’s.

EQUIPMENT:
Waterbath, Coplin jars, pH meter, Erlenmeyer flasks, graduated cylinders, filter paper.

TECHNIQUE:
Cut paraffin sections at 2 to 5µ.

QUALITY CONTROL:
A section of small intestine, appendix, or colon should be used as a positive control.


PRECAUTIONS:
Follow laboratory safety precautions: Labcoat, gloves, glasses, etc. Gloves are necessary to avoid absorption of dyes into skin. All acids are corrosive. Avoid  inhaling dye powders as synthetic dyes may cause neoplasms. Exercise caution when hamdling hot olutions.   Picric acid is unstable explosive if in the dry state, keep wet. Formalin is poison, may cause cancer. Fumes are hazardous, it is harmful if inhaled or absorbed through skin. Ferric chloride, phosphomolybdic acid and acetic acid are corrosive  Dispose of hazardous waste in appropriate receptacles.





SOLUTIONS:
SATURATED PICRIC ACID
Available from Polyscientific

BOUIN'S FLUID
Saturated Picric Acid                   75.0 ml
Formalin, Full Strength         25.0 ml
Glacial Acetic Acid                      5.0 ml

5% ALCOHOLIC HEMATOXYLIN
100% Ethanol                           100.0 ml
Hematoxylin                                5.0 gm

10% FERRIC CHLORIDE
Distilled Water                200.0 ml
Ferric Chloride                                20.0 gm

VERHOEFF'S IODINE
Distilled Water                   100.0 ml
Iodine                                   2.0 gm
Potassium Iodate                         4.0 gm

VERHOEFF'S ELASTIC TISSUE STAIN
5% Alcoholic Hematoxylin                22.0 ml
10% Ferric Chloride                       8.0 ml
Verhoeff's Iodine                         8.0 ml
Prepare fresh and use the same day.

1% BIEBRICH SCARLET
Distilled Water                100.0 ml
Biebrich Scarlet                           1.0 gm




1% ACID FUCHSIN
Distilled Water                 100.0 ml
Acid Fuchsin                            1.0 gm

BIEBRICH SCARLET-ACID FUCHSIN SOLUTION
1% Biebrich Scarlet                  45.0 ml
1% Acid Fuchsin                5.0 ml
Glacial Acetic Acid                    0.5 ml
Mix and filter to prevent red staining artifact.


PHOSPHOMOLYBDIC-PHOSPHOTUNGSTIC ACID SOLUTION
Distilled Water                 100.0 ml
Phosphomolybdic Acid            2.5 gm
Phosphotungstic Acid            2.5 gm

ANILINE BLUE SOLUTION
Distilled Water                100.0 ml
Aniline Blue                                  2.5 gm
Glacial Acetic Acid                       2.0 ml

1% GLACIAL ACETIC ACID
Distilled water              99.0 ml
Glacial Acetic Acid                       1.0 ml








STAINING PROCEDURE:
Preheat the Bouin’s in a 60° water bath for at least 10 minutes
1.      Hydrate slides to distilled water.
2.      Mordant slides in preheated Bouin's fluid in a 60° water bath for 5 minutes
3.         Wash in water until all yellow is removed.
4.      Verhoeff's Elastic Tissue Stain for 20 minutes.
5.      Rinse well in water.
6.      Differentiate in diluted 2% Ferric Chloride.  Elastic fibers should be black,  nuclei black, and other tissue elements dark gray.
7.      Rinse well in distilled water.
8.      95% Alcohol for 1 minute.
9.      Rinse in distilled water.
10.     Biebrich Scarlet-Acid Fuchsin Solution for 2 minutes.
11.     Rinse in distilled water.
12.     Phosphomolybdic-Phosphotungstic Acid for 20 minutes.                    USE FRESH.  DO NOT RINSE!
13.     Aniline Blue for 10 minutes. Rinse in distilled water.
14.     1% Glacial Acetic Acid for 3 minutes to blue.
15.     Rinse in distilled water.
16.     Dehydrate, clear, and mount.

RESULTS:

Nuclei and Elastic Fibers -                     Blue-Black to Black
Keratin and Muscle Fibers -                     Red
Collagen -                                      Blue

REFERENCES:     Sheehan, D. and Hrapchak, B., Theory and Practice of Histotechnology, 2nd       Edition.  St. Louis:  The C.V. Mosby Company, 1980.

________________________________________
From: histonet-bounces <@t> lists.utsouthwestern.edu [histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of karenadams <@t> comcast.net [karenadams <@t> comcast.net]
Sent: Monday, February 11, 2008 9:17 AM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] Verhoeff/ Trichome procedure

Does anyone have a procedure for Verhoeff's-Masson Trichrome they would be willing to share?

Thank you in advance :)

--
Karen Adams
Supervisor
Pathology Laboratories West
9303 Park West Blvd
Knoxville, TN 37923
(865) 690-2111 FAX (865) 691-1623
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