Effects of ethanol post-fixation on the histological, histochemical, and immunohistochernical analysis of murine bone specimens

Proper histological and histomorphometric assessment of bones requires acute visualization of cellular detail. Because fat is soluble in ethanol, adding an ethanol post-fixation step can help remove excess fat in tissue specimens, thus enhancing cellular visualization. However, the effects of adding this additional ethanol post-fixation step on bone-specific histochemical and immunohistochemical protocols have not yet been elucidated. Here, we describe the differences between using or not using an additional ethanol post-fixation step on six types of histochemical and immunohistochemical protocols. We assessed the effects in undecalcified tibiae sections routinely stained with hematoxylin and eosin, toluidine blue, and Von Kossa and found that addition of ethanol post-fixation enhanced specimen visualization for each of these stains. We examined the effect of ethanol post-fixation on enzymatic staining and found that alkaline phosphatase activity was equally preserved, whereas acid phosphatase activity was visualized more readily in specimens that underwent this treatment. Finally, the ethanol post-fixation improved antigen preservation in osteocalcin immunohistochemical labeling.