Hello everyone,
I’ve been encountering high background when immunostaining paraffin-embedded, fixed tissues that include both soft and bony components.
These specimens are periapical tissues obtained from human teeth during periapical surgeries and typically contain apical periodontitis lesions (granulomas/cysts), blood vessels, nerves, periodontal ligament, tooth, and bone. When bone is present on the slide, background staining is consistently high across multiple antibodies (typically neuronal and inflammatory cell markers such as NF-H, CGRP, PGP9.5, β-tubulin III, CD45, among others). In matched sections without bone, stained using the same antibodies and protocols, background is minimal to none.
Do you have any suggestions or tips for improving the signal-to-background ratio in these mixed soft/hard-tissue sections?
Thank you very much!
Hi @wtheodoro. This sounds tricky. Do you think it is related to the decalcification step or all of the tissue has been decalcified that you’re staining when you say background if bone is present? I would look up Patrick Mantyh lab papers perhaps? I actually did not notice in his papers anything special but he is an expert in molecular and cellular mechanisms of bone cancer pain and may be able to help you. You might also check Alejandro Almarza’s papers. He studies TMJ and has done a lot of histology on facial structures.
Anyone on Pain Researcher do this kind of histology?
-Becky
Hi Theodoro,
We routinely conduct IHC in bone and joint tissue within our lab although until recently mostly in mouse tissue. For mouse knee joint/bone tissue, the tissue processing, decalcification and IHC is pretty straightforward and the protocols outlined in prior publications by Pat Mantyh work pretty well for 20-micron thick sections. If you want to label thicker sections (80-120 micron) there are some considerations I can recommend if you are interested.
Working with human derived tissue poses several challenges including maintaining consistent and standardized methods for tissue processing, fixation and decalcification. We have found that signal to background ratio is improved if the samples are not postfixed for too extended of a time (approx. 24 hours) and if they are not paraffin embedded. You may want to reach out to Armen Akopian’s lab. They are conducting similar studies in patient TMJ samples as part of the Re-Join Consortium.
Chris