Abstract
Immunohistochemistry, or immunolabeling, is a key method for the identification of protein expression and localization. Successful detection relies on a low signal-to-noise ratio, which is affected greatly by antibody specificity as well as the staining protocol. Immunohistochemistry in the mouse is challenging, particularly in adult skeletal tissue, due to the need for long decalcification, high autofluorescence and high levels of endogenous peroxidase. Here, we describe a highly sensitive protocol for protein detection in decalcified paraffin-embedded sections from adult mouse skeletal tissue. By using four levels of amplification, this method allows for the identification of even low-abundance proteins.
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References
Shi ZR, Itzkowitz SH, Kim YS (1988) A comparison of three immunoperoxidase techniques for antigen detection in colorectal carcinoma tissues. J Histochem Cytochem 36:317–322. https://doi.org/10.1177/36.3.3278057
de Matos LL, Trufelli DC, de Matos MGL, da Silva Pinhal MA (2010) Immunohistochemistry as an important tool in biomarkers detection and clinical practice. Biomark Insights 5:9–20
Ward JM, Rehg JE (2014) Rodent immunohistochemistry. Vet Pathol 51:88–101. https://doi.org/10.1177/0300985813503571
Akkiraju H, Bonor J, Nohe A (2016) An improved immunostaining and imaging methodology to determine cell and protein distributions within the bone environment. J Histochem Cytochem 64:168–178. https://doi.org/10.1369/0022155415626765
Liu ES, Martins JS, Zhang W, Demay MB (2018) Molecular analysis of enthesopathy in a mouse model of hypophosphatemic rickets. Development 145:dev163519. https://doi.org/10.1242/dev.163519
Mathew SJ, Hansen JM, Merrell AJ et al (2011) Connective tissue fibroblasts and Tcf4 regulate myogenesis. Development 138:371–384. https://doi.org/10.1242/dev.057463
Stack EC, Wang C, Roman KA, Hoyt CC (2014) Multiplexed immunohistochemistry, imaging, and quantitation: a review, with an assessment of Tyramide signal amplification, multispectral imaging and multiplex analysis. Methods 70:46–58. https://doi.org/10.1016/J.YMETH.2014.08.016
Jiao Y, Sun Z, Lee T et al (1999) A simple and sensitive antigen retrieval method for free-floating and slide-mounted tissue sections. J Neurosci Methods 93:149–162. https://doi.org/10.1016/S0165-0270(99)00142-9
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Felsenthal, N., Zelzer, E. (2021). Immunofluorescent Staining of Adult Murine Paraffin-Embedded Skeletal Tissue. In: Hilton, M.J. (eds) Skeletal Development and Repair. Methods in Molecular Biology, vol 2230. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1028-2_19
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DOI: https://doi.org/10.1007/978-1-0716-1028-2_19
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Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-1027-5
Online ISBN: 978-1-0716-1028-2
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