To who this may concern ,
I am trying to stain cross-section of a nerve with toluidine blue for morphometric analysis of the section. I am trying to identify the fibre diameters and general characteristics. I am using 5-6 micrometer sections which have been embedded in paraffin. I have not been able to get a good technique for the stain due to my sections not retaining the blue color. In other words, my sections turn pink/purple after staining and I am not able to analyze the tissue as the color is very faint. Any suggestions or protocols for this stain ?
Mehran Hosseini Thank you for your response. Will this staining technique of luxe blue enable me to qualitatively analyze the section with regards to the number of fibers and the diameters of the fibers within the section? Is this a more efficient method than the toluidine blue approach?
Dear Brigette,
The LFB technique usually uses for myelin integrity assessment and cryostat (frozen sections) sections are preferred for this technique. However, there is evidence regarding its efficiency in the paraffin-embedded sections. In my view, the LFB as a simple method might be useful for counting and evaluating axonal quality. I recommend using thick sections (20 um).
Good luck,
Mehran
Toluidinblue is a metachromatic dye. Binding to "chromotrops" it turns reddish. Do you rinse your slides in acidified water or tapwater after staining? When the stained tissue is dehydrated through ethanol the blue colour should appear again, because metachromasia needs an aequous medium. The exception is so called "true metachromasia", that can be found with strongly sulfated proteoglycans. (eg. in mastcells)
If your tissue is full of such proteoglykans, you will get a red-violet result.
Gudrun Lang I do rinse my slides in tap-water directly after staining according to my protocol. I am now changing this for DI water. Even after the dehydration , my slides tend to remain a red/ pink color.
Mehran Hosseini Thank you for your advice. I am looking into it , it seems like it will be effective with a combination of the cresyl violet staining also! There are many papers validating this method and well as the LB staining solely. My only concern is that my research calls for sections of 4-6 micrometers thin.
Another try: prolong toluidinblue staining. minimize rinsing.
Are the slides blue at any time after staining?
Maybe this could also help: rinse the slides in DI after staining, blot off excessive water and let them dry for half a minute. Then go directly and very short (1-2 dips) in abs. ethanol and then in xylene.
Has anyone other success with this dye in your lab? Sometimes dye-batches are of minor quality or have impurities.
There are different protocols for "over-all" staining with toluidinblue and for staining eg. mastcells. Over-all stains use alkaline solutions. (with borax)
Which recipe do you use?
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