TY - JOUR
T1 - Tissue-specific Fixation Methods Are Required for Optimal In Situ Visualization of Hyaluronan in the Ovary, Kidney, and Liver
AU - Rowley, Jennifer E.
AU - Rubenstein, Gillian E.
AU - Manuel, Sharrόn L.
AU - Johnson, Natalie L.
AU - Surgnier, Jordan
AU - Kapitsinou, Pinelopi P.
AU - Duncan, Francesca E.
AU - Pritchard, Michele T.
N1 - Funding Information:
We would like to thank Jing Huang for her technical assistance in ovarian tissue sectioning. We also thank board certified pathologists Dr. Tim Fields and Dr. Katie Dennis for their assistance in reviewing and evaluating the kidney and liver histology, respectively. Finally, we would like to acknowledge the late Dr. Mark E. Lauer for the Hyaluronan Binding Protein (HABP) Fluorescent Staining Protocol that he developed as part of the Cleveland Clinic Program of Excellence in Glycoscience?Hyaluronan Matrices in Vascular Pathologies (award number P01HL107147) that was modified for this study. The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by a Capstone Grant from the Histochemical Society (to GER), R01 HD093726 (to FED and MTP) from the Eunice Kennedy Shriver National Institute of Child Health & Human Development, R01 DK115850 (to PPK) from the National Institute of Diabetes and Digestive and Kidney Diseases, Northwestern University?s Center for Reproductive Science Marcia L. Storch Scholarship Fund for Undergraduate Women (to GER), and the Society for Toxicology Summer Undergraduate Research Fellowship (to NLJ). The KUMC Histology Core was supported by the P30 HD002528 (Kansas IDDRC).
Publisher Copyright:
© The Author(s) 2019.
PY - 2020/1/1
Y1 - 2020/1/1
N2 - Hyaluronan (HA) is a ubiquitous component of the extracellular matrix. The spatial-temporal localization of HA can be visualized in situ using biotinylated HA binding proteins (HABPs). This assay is sensitive to fixation conditions, and there are currently no best practices for HA detection. Thus, the goal of this study was to optimize fixation conditions for visualizing HA in the ovary, kidney, and liver through analysis of six commonly used fixatives for HA detection: Bouin’s Solution, Carnoy’s Solution, Ethanol-Formalin-Glacial Acetic Acid (EFG), Histochoice, Modified Davidson’s Solution, and 10% Neutral Buffered Formalin. Organs were harvested from CB6F1 mice and fixed with one of the identified fixatives. Fixed organs were sectioned, and the HABP assay was performed on sections in parallel. Hematoxylin and eosin staining was also performed to visualize tissue architecture. HABP signal localization and intensity varied between fixatives. EFG and Carnoy’s Solution best preserved the HA signal intensity in the ovary and liver, showing HA localization in various sub-organ structures. In the kidney, only Modified Davidson’s Solution was less than optimal. Our findings demonstrate that fixation can alter the ability to detect HA in tissue macro- and microstructures, as well as localization in a tissue-specific manner, in situ.
AB - Hyaluronan (HA) is a ubiquitous component of the extracellular matrix. The spatial-temporal localization of HA can be visualized in situ using biotinylated HA binding proteins (HABPs). This assay is sensitive to fixation conditions, and there are currently no best practices for HA detection. Thus, the goal of this study was to optimize fixation conditions for visualizing HA in the ovary, kidney, and liver through analysis of six commonly used fixatives for HA detection: Bouin’s Solution, Carnoy’s Solution, Ethanol-Formalin-Glacial Acetic Acid (EFG), Histochoice, Modified Davidson’s Solution, and 10% Neutral Buffered Formalin. Organs were harvested from CB6F1 mice and fixed with one of the identified fixatives. Fixed organs were sectioned, and the HABP assay was performed on sections in parallel. Hematoxylin and eosin staining was also performed to visualize tissue architecture. HABP signal localization and intensity varied between fixatives. EFG and Carnoy’s Solution best preserved the HA signal intensity in the ovary and liver, showing HA localization in various sub-organ structures. In the kidney, only Modified Davidson’s Solution was less than optimal. Our findings demonstrate that fixation can alter the ability to detect HA in tissue macro- and microstructures, as well as localization in a tissue-specific manner, in situ.
KW - extracellular matrix
KW - hyaluronan binding protein assay
KW - preservation—biological
KW - staining/labeling
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U2 - 10.1369/0022155419884879
DO - 10.1369/0022155419884879
M3 - Article
C2 - 31714169
AN - SCOPUS:85075194007
VL - 68
SP - 75
EP - 91
JO - Journal of Histochemistry and Cytochemistry
JF - Journal of Histochemistry and Cytochemistry
SN - 0022-1554
IS - 1
ER -