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Identifying cell populations and their proteins within bone is necessary for understanding bone biology. Immunolabeling is one approach used to visualize proteins in tissues. Efficient immunolabeling of bone samples often requires decalcification, which may lead to changes in the structural morphology of the bone. Recently, methyl-methacrylate embedding of non-decalcified tissue followed by heat-induced antigen retrieval has been used to process bone sections for immunolabeling. However, this technique is applicable for bone slices below 50-\u00b5m thickness while fixed on slides. Additionally, enhancing epitope exposure for immunolabeling is still a challenge. Moreover, imaging bone cells within the bone environment using standard confocal microscopy is difficult. Here we demonstrate for the first time an improved methodology for immunolabeling non-decalcified bone using a testicular hyaluronidase enzyme-based antigen retrieval technique followed by two-photon fluorescence laser microscopy (TPLM) imaging. This procedure allowed us to image key intracellular proteins in bone cells while preserving the structural morphology of the cells and the bone.<\/jats:p>","DOI":"10.1369\/0022155415626765","type":"journal-article","created":{"date-parts":[[2016,1,11]],"date-time":"2016-01-11T20:08:10Z","timestamp":1452542890000},"page":"168-178","update-policy":"https:\/\/doi.org\/10.1177\/sage-journals-update-policy","source":"Crossref","is-referenced-by-count":11,"title":["An Improved Immunostaining and Imaging Methodology to Determine Cell and Protein Distributions within the Bone Environment"],"prefix":"10.1369","volume":"64","author":[{"given":"Hemanth","family":"Akkiraju","sequence":"first","affiliation":[{"name":"Department of Biological Sciences, University of Delaware, Newark, Delaware (HA, JB, AN)"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jeremy","family":"Bonor","sequence":"additional","affiliation":[{"name":"Department of Biological Sciences, University of Delaware, Newark, Delaware (HA, JB, AN)"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Anja","family":"Nohe","sequence":"additional","affiliation":[{"name":"Department of Biological Sciences, University of Delaware, Newark, Delaware (HA, JB, AN)"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"179","published-online":{"date-parts":[[2015,12,30]]},"reference":[{"key":"bibr1-0022155415626765","doi-asserted-by":"publisher","DOI":"10.1002\/jor.22752"},{"key":"bibr2-0022155415626765","doi-asserted-by":"publisher","DOI":"10.1385\/1592594174"},{"key":"bibr3-0022155415626765","unstructured":"Bancroft JD, Stevens A (1982). 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