2.5. Histology and Macrophage (F4/80), Osteoclast (TRAP), and Osteoblast (OCN) Staining

After microCT scanning of the fixed right forearm, bones were decalcified in 14% EDTA then placed in 30% sucrose overnight. Forearms were cryo-embedded (TissueTek O.C.T) and sectioned transversely at 16–18 μm. The position of the callus was guided by the microCT scan, as well as by visual inspection of the sections. Hematoxylin and eosin (H&E) staining was performed using routine protocol.

To determine if macrophages were present in the callus, immunostaining for F4/80 was performed with a rat anti-mouse F4/80 clone CI:A3–1 antibody (Bio-Rad MCA497RT and Abcam ab6640). Briefly, for the antibody sourced from Bio-Rad (MCA497RT), samples were blocked with 5% heat-inactivated donkey serum in 1x PBS for two hours, then stained with F4/80 rat anti-mouse antibody at 1:100 dilution in blocking solution and visualized with a Cy3 goat anti-rat secondary antibody (Thermo Fisher A1434) with a 1:500 dilution. Sections were cover-slipped with Prolong Gold with DAPI and imaged on a Nikon microscope with a fluorescent source. When Abcam ab6640 was used, sections were treated with trypsin for antigen retrieval, followed by blocking with Background Sniper (Biocare Medical BS966). The primary antibody was diluted at 1:250 in DaVinci Green Diluent (Biocare Medical PD900) and visualized with an Alexa Fluor 647 goat anti-rat secondary antibody (Biolegend 405416) using a 1:200 dilution. Sections were cover-slipped with Prolong Gold with DAPI and imaged on a Leica Thunder DMi8 microscope. Analysis was done using ImageJ (National Institutes of Health, NIH).

Osteoclasts were visualized following a tartrate-resistant acid phosphatase (TRAP) stain protocol as previously described (25) with an additional pre-incubation in 0.2 M tris hydrochloride for 1 hour at 37°C. Color variations are hematoxylin dependent.

For osteoblast detection, osteocalcin (OCN) staining was performed. Slides were subjected to antigen retrieval by warmed (37°C) proteinase K (Thermo Fisher AM2546) in TEN buffer followed by several washes with tris buffered saline (TBS). Next, blocking was performed with a 10% heat-inactivated fetal bovine serum and 10% heat-inactivated normal goat serum in TBS for one hour at room temperature. Sections were exposed to the primary antibody (rat anti-mouse osteocalcin 1:300, Thermo Fisher PA5–78870) for two hours at room temperature, followed by washes, and exposure to the secondary goat anti-rabbit Alexa Fluor 680 (Thermo Fisher A21076) antibody in a 1:200 dilution. Slides were cover-slipped with Prolong Gold with DAPI. Images were taken on a Leica Thunder DMi8 and analyzed with ImageJ.