Optimization of immunohistochemical detection of collagen type II in osteochondral sections by comparing decalcification and antigen retrieval agent combinations |
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Authors: | Soosai Manickam Amirtham Ozlem Ozbey Upasana Kachroo Boopalan Ramasamy Elizabeth Vinod |
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Institution: | 1. Department of Physiology, Christian Medical College, Vellore, 632002 Tamil Nadu, India;2. Department of Histology and Embryology Campus, School of Medicine, Akdeniz University, Antalya, 07070 Turkey;3. Department of Orthopaedics, Royal Darwin Hospital, 105 Rocklands Drive, Tiwi NT, 0810 Australia;4. Department of Physiology, Christian Medical College, Vellore, 632002 Tamil Nadu, India
Centre for Stem Cell Research, Christian Medical College, Vellore, 632002 Tamil Nadu, India |
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Abstract: | Bone containing tissues such as osteochondral joint are resistant to routine tissue processing, therefore require decalcification. This technique causes removal of mineral salts, but in the process may macerate the organic tissue, hence the need for tissue fixation. Such severe processing demands careful antigen retrieval to necessitate optimal staining. The aim of our study was to compare five different antigen retrieval protocols (heat retrieval and protein digestion) following decalcification of rabbit knee joints using two different techniques (20% formic acid and 10% ethylenediamine-tetra acetic acid: EDTA). Osteochondral sections were compared based on time required for decalcification, ease of sectioning, morphological integrity using HE staining and antigen preservation (Collagen type II) using immunohistochemistry. The two decalcification solutions did not impair the tissue morphology and ease of sectioning. Joints processed with formic acid decalcified four times faster than EDTA. Among the five antigen retrieval approaches, maximal collagen II uptake with minimal nonspecific staining was found with protein digestion (pronase and hyaluronidase) in both formic acid and EDTA sections. For osteo-chondral sections, we recommend using 10% EDTA for decalcification and pronase plus hyaluronidase for antigen retrieval if maintaining tissue morphology is crucial, whereas if time is of the essence, 20% FA with pronase plus hyaluronidase is the faster option while still preserving structural integrity. Clin. Anat. 33:343–349, 2020. © 2019 Wiley Periodicals, Inc. |
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Keywords: | imaging decalcification immunohistochemistry collagen II cartilage bone tissue |
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