Picrosirius Red Staining Procedure

Purpose:
This standard operating procedure (SOP) establishes the standard for staining collagen using the Picrosirius Red staining procedure.

Reagents and Materials:

Reagents

0.2% Phosphomolybdic Acid
Phosphomolybdic Acid 2gm
Distilled water 1000ml


Saturated Picric Acid
Picric Acid 6.0gm
Distilled water 200ml

Picrosirius Red F3BA
Saturated Picric Acid 400ml
Sirius Red 0.4gm

.01 N Hydrochloric Acid:
Hydrochloric Acid 10ml
Distilled water 992ml

Materials
Tissue slides and control slides for special stain requested.
Microwave to bake slides.
Staining rack, 20 slide capacity.
Coverslips and synthetic mounting media.

Hazards and Precautions:
See Material Safety Data Sheets.

Sample Preparations:
Paraffin tissue section slides cut at 4 microns.

Calibration and Standardization:
A tissue section slide containing bone is stained with each procedure.

Procedure:

1. Deparaffinize and hydrate to distilled water.

2. .0.2% Phosphomolybdic Acid - 2 minutes.

3. Distilled water rinse.

4. Picrosirius Red - 110 minutes.

5. 0.01N Hydrochloric Acid - 2 minutes.

6. 70% Ethanol - 45 seconds.

7. Dehydrate, clear and coverslip.

Results:
Stains fibrillar type1 and type3 collagen.
Collagen - red
Type 1 - yellow
Type 3 - green

Figure 1. Positive control bone and adjacent muscular artery. Picrosirius red, nonpolarized left, polarized right with birefringent collagen types I & III.

Figure 2. Interstitial and perivascular myocardial fibrosis, Picrosirius red, nonpolarized left, polarized right with birefringent collagen types I & III.

Effective: July 1, 2004
Cherie Chapman
James R. Turk

References:

Carrasco FH, Montes GS, Krisztan RM, Shigihara KM, Carneiro J, Junqueira LC. Comparative morphologic and histochemical studies on the collagen of vertebrate arteries. Blood Vessels. 1981;18:296-302.

Crisby M, Nordin-Fredriksson G, Shah PK, Yano J, Zhu J, Nilsson J. Pravastatin treatment increases collagen content and decreases lipid content, inflammation, metalloproteinases, and cell death in human carotid plaques: implications for plaque stabilization. Circulation. 2001;103:926-33.

Noorlander ML, Melis P, Jonker A, Van Noorden CJ. A quantitative method to determine the orientation of collagen fibers in the dermis. J Histochem Cytochem. 2002;50:1469-74.

Pick R, Janicki JS, Weber KT. Myocardial fibrosis in nonhuman primate with pressure overload hypertrophy. Am J Pathol. 1989; 135:771-781.

Wadsworth MP, Sobel BE, Schneider DJ, Taatjes DJ. Delineation of the evolution of compositional changes in atheroma. Histochem Cell Biol. 2002;118:59-68.

Weber KT, Pick R, Silver MA, Moe GW, Janicki JS, Zucker IH, Armstrong PW. Fibrillar collagen and remodeling of dilated canine left ventricle. Circulation. 1990;82:1387-1401.