Hello all,
I am having some serious trouble staining resin embedded rat bone using basic fuchsine and toluidine blue. I have tried many different protocols but it seems like the bone matrix does not really stain on either of them.
The protocols I have tried are:
- Dissolve 730 mg of toluidine blue O and 270 mg of basic fuchsin together in 100 mL of 30% alcohol. Staining for 5 min without heat or incubating for 5min on 60degrees.
- Same ratio of toluidine blue O and basic fuchsine was used but not mixed together. Toluidine blue O was diluted with distilled water. First applied toluidine blue for 5 min with and without heat as previous stated. Thereafter tried first incubating toluidine blue for 5 min on 60degrees and thereafter 1h before adding basic fuchsine for 5min and then 1h.
--Tried added borax to the separate solutions to see if it helped penetrate the resin. It helped indeed with the toluidine blue (double the amount of borax was added in relation to toluidine blue) , but no difference was seen on basic fuchsine. Tried adding borax (0.1M v/v 1:5) just before staining to the basic fuchsine solution and incubating for 30sek on 100degrees, no real difference there.
After every staining I wahs the slide with distilled water.
Am I missing something vital? The staining seem to attach on both nuclei and cell matrix, just not on bone or collagen where it suppose to stain a dark purple.
Would greatly appreciate your help!
Thank you in advance.
Regards,
Elina
Hello,
In order to effectively help you could you please give some additional information?
Which resin did you use?
It looks like you have stained thin ground sections. Is that correct?
If we are talking about undecalcified thin ground sections, you could use hydrogen peroxide to etch the surface and acetic acid to slightly decalcify the surface before staining. Both methods improve the staining intensity.
Did you apply staining protocols for paraffin histology? These usually don't work in resin embedded specimens. It needs specialized staining protocols adapted to the resin you use.
Stefan Tangl Of course!
I used Technovit 7100 kit which is an glycol methacrylate based resin.
The slides are thinly sectioned to around 40-55um and later polished and the bones have not been decalcified. How do you go about using hydrogen peroxide and acetic acid? Would wiping the slides with the solution suffice?
No, the protocols I have attempted are for resin embedded tissue! Is there more prep work that has to be applied to the slides before staining for the staining to work more efficiently?
Etching and surface decalcification is done as follows in our lab.
Wiping is not enough, you have to immerse the specimens in a container filled with the reagents.
1) Etching the surface by immersing the slide in 30% hydrogen peroxide H2O2: 3 minutes
Move either the solution or the specimen slightly during etching.
Rinse in tap water (2x) & dry specimen with dry smooth cloth
2) Decalcifying the surface immersing the slide in 10% acetic acid (CH3COOH): 30 seconds
Rinse in tap water (2x) & dry specimen with dry smooth cloth
But we use Technovit 7200 VLC. You might have to adapt the duration of the proceedures to Technovit 7100, probably make them longer. We stained specimens embedded in Technovit 9100 once and the staining was much weaker than with 7200. Seems to be a similar problem as yours. You could also try to increase the concentration of the reagents stepwise to inhance the effect of etching and surface decalcification.
Concerning prep work:
I suppose you have mounted the section on glass or plexiglas slides? It gives a much clearer picture with fewer scratches if you polish the section on both sides before mounting.
You can download a pdf with preparation and staining procedures (for Technovit 7200) from the following link:
https://www.dropbox.com/s/2w73lwrkytnauj5/donath_preparation_of_histologic_sections.pdf?dl=0
Stefan Tangl Thank you so much for such a detailed response! Since no one in my lab have any experience with hard histology I really appreciate your insight and expertise. I tried your protocol and it really improved the staining quality and stained the bone. Thanks again!!
I am glad to hear it worked. If you have any other questions concerning hard tissue histology, please feel free to contact me.
And if you allow me a little bit of self-advertising:
We have a course of Research Acadamy of the Osteology Foundation here in our Lab in Vienna from 2-4 November 2020.
https://www.osteology.org/education/research-academy/
It covers hard tissue research from histology to morphometry to micoCT analysis.
If you or members of your group want to stay in hard tissue research, it might be interesting to participate.
Stefan Tangl Sounds super interesting! I have forwarded the link to my supervisor and she was definitely interested.
I have run into a smaller issue with my toluidine blue stain, it does not penetrate the cartilage as well as i would like to. Is this something you have experience in as well?
Sorry, we have never used toluidine blue on cartilage, as I know it only as a typical and very effective bone stain. You could try to increase (double?) the application time of 30% hydrogen peroxide H2O2 for etching the surface.
This mostly influences soft tissue staining and could improve cartilage staining. But you have to expect that other soft tissues will stain more intensely too, or even overstain.
There apear to be experts for cartilage staining with toluidine blue in Aarhus. I am sure they know more about it than I do:
https://journals.sagepub.com/doi/full/10.1177/1947603518764262?url_ver=Z39.88-2003&rfr_id=ori:rid:crossref.org&rfr_dat=cr_pub%3dpubmed
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