Hi everyone.
Does exist a protocol to toluidine staining in monolayer cells? Or someone have a simple protocol to it?
Dear Bruna,
The following is the required protocol:
Toluidine Blue Staining Protocol for Mast Cells
NovaUltra Special Stain Kits
Description: Mast cells are found in the connective tissue and their cytoplasm contains granules (metachromatic) composed of heparin and histamine. Toluidine blue should stain mast cells red-purple (metachromatic staining) and the background blue (orthochromatic staining). Metachromasia, tissue elements staining a different color from the dye solution, is due to the pH, dye concentration and temperature of the basic dye. Blue or violet dyes will show a red color shift, and red dyes will show a yellow color shift with metachromatic tissue elements.
Fixation: 10% Formalin.
Sections: paraffin sections at 5 um.
Solutions and Reagents:
Toluidine Blue Stock Solution:
Toluidine blue O (Sigma) --------------- 1 g
70% alcohol ------------------------------ 100 ml
Mix to dissolve.
Sodium Chloride (1%):
Sodium chloride --------------------------- 0.5 g
Distilled water ----------------------------- 50 ml
Mix to dissolve (make this solution fresh each time). Adjust pH to 2.0~2.5 using glacial acetic acid or HCl.
Toluidine Blue Working Solution (pH 2.0~2.5):
Toluidine blue stock solution -------------- 5 ml
1% Sodium chloride, pH 2.3 --------------- 45 ml
Mix well. The pH should be around 2.3 and less than 2.5
Make this solution fresh and discard after use. pH higher than 2.5 will make staining less contrast.
Procedure:
1. Deparaffinize and hydrate sections to distilled water.
2. Stain sections in toluidine blue working solution for 2-3 minutes.
3. Wash in distilled water, 3 changes.
4. Dehydrate quickly through 95% and 2 changes of 100% alcohol (10 dips each since stain fades quickly in alcohol).
5. Clear in xylene or xylene substitute, 2 changes, 3 minutes each.
6. Coverslip with resinous mounting medium.
Results:
Mast cells --------------------------- violet/red purple.
Background ------------------------- blue.
Find Images
http://www.ihcworld.com/_protocols/special_stains/toluidine_blue.htm
Hoping this will be helpful,
Rafik
Dear Bruna,
The following is the required protocol:
Toluidine Blue Staining Protocol for Mast Cells
NovaUltra Special Stain Kits
Description: Mast cells are found in the connective tissue and their cytoplasm contains granules (metachromatic) composed of heparin and histamine. Toluidine blue should stain mast cells red-purple (metachromatic staining) and the background blue (orthochromatic staining). Metachromasia, tissue elements staining a different color from the dye solution, is due to the pH, dye concentration and temperature of the basic dye. Blue or violet dyes will show a red color shift, and red dyes will show a yellow color shift with metachromatic tissue elements.
Fixation: 10% Formalin.
Sections: paraffin sections at 5 um.
Solutions and Reagents:
Toluidine Blue Stock Solution:
Toluidine blue O (Sigma) --------------- 1 g
70% alcohol ------------------------------ 100 ml
Mix to dissolve.
Sodium Chloride (1%):
Sodium chloride --------------------------- 0.5 g
Distilled water ----------------------------- 50 ml
Mix to dissolve (make this solution fresh each time). Adjust pH to 2.0~2.5 using glacial acetic acid or HCl.
Toluidine Blue Working Solution (pH 2.0~2.5):
Toluidine blue stock solution -------------- 5 ml
1% Sodium chloride, pH 2.3 --------------- 45 ml
Mix well. The pH should be around 2.3 and less than 2.5
Make this solution fresh and discard after use. pH higher than 2.5 will make staining less contrast.
Procedure:
1. Deparaffinize and hydrate sections to distilled water.
2. Stain sections in toluidine blue working solution for 2-3 minutes.
3. Wash in distilled water, 3 changes.
4. Dehydrate quickly through 95% and 2 changes of 100% alcohol (10 dips each since stain fades quickly in alcohol).
5. Clear in xylene or xylene substitute, 2 changes, 3 minutes each.
6. Coverslip with resinous mounting medium.
Results:
Mast cells --------------------------- violet/red purple.
Background ------------------------- blue.
Find Images
http://www.ihcworld.com/_protocols/special_stains/toluidine_blue.htm
Hoping this will be helpful,
Rafik
Dear Bruna,
Usually, we used this protocol for staining after MSCs differentiation:
1. washed the cells twice with PBS and fixed them with formaldehyde (4%) at least for 30 min.
2. after fixation wash twice and then add the Toluidine blue for 30 minutes.
3. after staining wash the slides for 5 min with running distilled water. Repeat the washing three times until the water is clean.
Toluidine blue preparation: dissolve the Toluidine blue in distilled water (0.1 g of Toluidine blue in 100 ml of distilled water). And check pH of the solution, it is very important.
Hope, it will be useful
Thank you very much for the answers!
The protocols were very helpful!
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