Does anyone have some experience in staining macrophage in periodontal tissue, especially in periodontally-infected (i.e., periodontitis) tissue in mice? Growing attention is paying on macrophage in periodontitis. However, it seems too few macrophages exist in periodontitis, let alone in the tiny gingiva in mice. If I hope to immunostain macrophages in paraffin-embedding jaw from mice, what are the notes during decalcification, tissue processing and immunohistochemistry?
I hope an attached manuscript help you to perform your experiments successfully.
Hy, try to decalcificate the tissue max 0.5 cm thick for 2-5 days (after 24-48 h in 10% neutral-buffered formalin) with
Solution A:
Sodium citrate 50 gr.
Distilled water 250 ml
Solution B:
Formic acid 90% 125 ml
Distilled water 125 ml
Mix solution A and B in equal portions for use
Dear Ting,
First be sure that the tissues are well fixed (10% buffered formalin/5% paraformaldehyde 0.1 M phosphate (pH 7.3) and decalcified in EDTA (pH 7.0).
An easy approach to begin with is the classical histochemical staining for acid-phosphatase which will stain all osteoclasts and macrophages. In an adjacent section by adding tartrate to the substrate you block macrophage staining and only osteoclasts remain stained. Osteoclasts furthermore are larger, located alt teh boen margin and mostly multinucleated
You could try antibody to F480 which in mice is a macrophage marker but it may require antigen retrieval (either hot citrate buffer pH 6.0 or hot EDTA pH 9.0 buffer. I amnot familiar with macrophage markers but there should be molecules that are specific for macrophages.
To distinguish between macrophages /osteoclasts another approach is to stain for
v-H-ATPase with anti-v-H-ATpase (Tcirg1, Bone 50, pg 908, 2012) or carbonic anhydrase 2; by combining TRAP staining and immunostaining for v_H_ATPase on the same sections. Only osteoclasts will be stained both with TRAP and v-H_Atpase or CAR2.
Good luck
Ton Bronckers
Dear Stefano Rocca, thank you for your kind advice in decalcification procedure! I wonder if the decalcification method using formic acid will be good for immunohistochemistry? Unfortunately, if so, I have decalcified the mice jaw with 10% EDTA (pH=7.0, buffered by Tris) for nearly one month.
Dear Antonius Bronckers, Thank you for your so kind advice in detail ! I'm itching to try the classical TRAP staining method in periodontal tissue. Could you give me a TRAP protocol for parraffin-embedding jaw from mice, since very few articles have descripted this condition in detail? Your suggestions on distinguishing macrophage from osteoclast in periodontal tissue is noticeable and very useful. But I wonder if the TRAP kit will stain any cells other than macrophage and osteoclast, e.g., if any, mast cell or plasma cell? As you said, osteoclast is multicleated (>3 nuleus could be seen as a threshold ) which might be excluded to count macrophage in periodontal tissue? Strictly speaking, there are no specific but more representative markers for macrophage, such as F4/80 and CD68, with which I would try. According to your experience, how many macrophages-like cells could be stained in subepithelial connective tissue?
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