PRINCIPLE: Masson’s Trichrome staining technique is used for the detection of collagen fibers in tissues.
The samples are formalin-fixed, paraffin-embedded sections, or frozen sections (the video has shown qualification for non-frozen samples).
Weigert’s Hematoxylin, an iron hematoxylin dye is used to stain the nuclei, and this dye is resistant to decolorization by acidic staining solutions.
Biebrich scarlet-acid fuschin solution stains all the acidic tissues such as the cytoplasm, muscle, and collagen.
Phosphomolybdic or phosphotungstic acid is used as a decolorizing agent, making the Biebrich Scarlet-acid fuschin to diffuse out of the collagen fibers, which causes the muscle cells to stain red.
Aniline blue stains the collagen along which 1% acetic acid is added to show a difference in the tissue sections. The collagen fibers stain blue and the nuclei stains black, with a red background.
Fixation: 10% Neutral Buffered Formalin
Section: Paraffin section at 5-6micron
NOTE: This video has mentioned how to do the quantification of the stained area using
ImageJ Software in Masson's Trichome staining.
Reagents and Preparation
Weigert’s Iron Hematoxylin Solution
Stock Solution A
§ Hematoxylin - 1g
§ 95% alcohol -100ml
Stock solution B
§ 29% Ferric chloride in water- 4ml
§ Distilled water -95ml
§ Concentrated Hydrochloric acid -1ml
Equal parts of stock solutions A and B are mixed for use. The mixture is only stable for not more than 3 months
Biebrich Scarlet-Acid Fuschin Solution
§ 1% aqueous Biebrich Scarlet- 90ml
§ 1% aqueous Acid fuschin -10 ml
§ Glacial acetic acid -1 ml
Phosphomolybdic-Phosphotungstic Acid Solution
§ 5% Phosphomlybdic acid -25ml
§ 5% Phosphotungstic acid -25ml
Aniline Blue Solution
§ Aniline blue- 2.5g
§ Glacial acetic acid -2ml
§ Distilled water- 100ml
1% Acetic Acid Solution
§ Glacial acetic acid -1ml
§ Distilled water- 99ml
Procedure
1. De-paraffinize the section.
2. Wash in distilled water.
3. Stain with Weigert’s iron hematoxylin solution for 10 minutes.
4. Rinse the stain with running tap water for 5-10 minutes.
5. Wash in distilled water.
6. Stain with the Beibrich-Scarlet Acid Fuschin solution for 5-10 minutes.
7. Wash in distilled water.
8. Differentiate in the phosphomolybdic-phosphotungstic acid solution for 10-15 minutes or until the collagen loses its red color.
9. Transfer the stained section directly to aniline blue solution and stain for 5-10 minutes.
10. Rinse the stained section briefly in distilled water and differentiate it with 1% acetic acid solution for 1-2 minutes.
11. Wash in distilled water.
12. Quickly dehydrate through 95% ethyl alcohol.
13. Clear in xylene.
14. Mount with a mounting medium.
Result
§ Collagen fiber stains- blue.
§ Nuclei stains -black.
§ Muscles, cytoplasm, and keratin stain -red.
NOTE: Microwave procedure
1. Deparaffinize slides and hydrate to deionized water.
2. Place slides in 40 ml Weigert’s Iron Hematoxylin Solution, contained in a plastic Coplin jar, then microwave for 5 seconds at 600- 800 watts.
3. Rinse well in running tap water for 30 seconds to 1 minute.
4. Blue in Working Scott’s Tap Water Substitute at room temperature.
5. Rinse well in running tap water.
6. Place slides in Biebrich Scarlet-Acid Fuchsin Solution contained in a plastic Coplin jar, Microwave at 600 watts for 20 seconds.
7. Rinse quickly in several changes of deionized water.
8. Place slides in Phosphotungstic-Phosphomolybdic Acid Solution contained in a plastic Coplin jar, Microwave for 20 – 40 seconds. Immediately remove the slides and rinse in several changes of deionized water.
9. Place slides in Aniline Blue Solution contained in a plastic Coplin jar.
10. Rinse well in deionized water.
11. Place slides in 1% Acetic Acid for 30 seconds to 1 minute at room temperature.
12. Rinse slides, dehydrated through alcohol, and clear and coverslip.
Hi. I’m Writer of Researchsop.com. ’ ’ Please share these SOPs to all concern pharma people for their development. I like to fullfill the need of curious people. These things inspire me to make things looks better.
0 comments:
Post a Comment