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?