4.4. Histology and Immunohistochemistry
SEPARATION
Fixed bone tissue samples were decalcified in Osteofast 2 solution (Biognost, Zagreb, Croatia), embedded in paraffin wax and cut into 5 μm thick tissue sections. Tissue sections were then stained with hematoxylin-eosin and examined under a light microscope with a video camera. For histomorphometric analysis, photomicrographs at 10× magnification were transferred to ImageJ v1.53 software (NIH, For immunohistochemical analysis, previously prepared 5 μm thick tissue sections were deparaffinized, dehydrated, and then treated with 0.3% HQuantification of immunohistochemical staining was analyzed on previously recorded photomicrographs using the computer program cellSense v3.2. Photomicrographs taken at 200× magnification were subjected to intensity separation, its conversion to black and white, and background signal subtraction. The regions of interest were determined and placed on the obtained image display, and the final result was shown as the mean value of the color intensity—mean gray value. Histological and immunohistochemical examination were blindly performed by one investigator (O.C.P.).