Identification and Quantitation of Bone Marrow Adipocytes by Immunostaining
Chen, Yimeng (2022)
Chen, Yimeng
2022
Julkaisun pysyvä osoite on
https://urn.fi/URN:NBN:fi-fe2022060242724
https://urn.fi/URN:NBN:fi-fe2022060242724
Tiivistelmä
Bone marrow adipocytes (BMAds) have been found to impact other cell populations inside the marrow and they can influence the entire body, but the role of adipocytes in bone physiology has been poorly understood. BMAds can be identified in histological samples. They are commonly identified as unstained white and round objects in sections when using the hematoxylin and eosin (H&E) staining method. There is a need for additional specific detection by immunohistochemistry (IHC) to discern microvasculature from adipocytes. The aim of the study was to develop and optimize a method for the identification and quantitation of BMAds in formalin-fixed paraffin- embedded (FFPE) long bone tissue sections by immunostaining of adipocyte-specific protein perilipin-1. Secondly, our aim was to apply the IHC method and a semi-automated ImageJ script to identify and quantitate BMAds in rat long bone sections under two experimental conditions. The staining protocol was optimized at the beginning. Various staining parameters were optimized to immunobiologically stain rat perilipin-1 in FFPE long bone sections. After that, a workflow of image analysis was developed. Quantitative analysis was carried out by using segmentation-based approaches utilizing common bioimage analysis software. μCT was used to obtain volumetric bone mineral density (BMD) and structural parameters on bone quantity and phenotype. Tibiae were collected from rats supplemented with high-fat diet and control rats to test and verify the optimized protocol. The final optimized protocol was established from all parameters (deparaffinization, antigen retrieval, permeabilization, blocking, antibody concentration and autofluorescence reduction). The optimized semi-automated quantitation method was comparable to the manual counting. (R=0.959)
The number of BMAds was typically 300-500 /mm2 in the bone marrow region and the size range of cells was about 700 to 1000 μm2. Our optimized IHC method brings improvement over H&E as it can positively stain BMAds. It can be applied to histological bone tissue sections. An improved quantitation analysis based on this IHC method was presented by the end. Pilot studies from the μCT from different groups showed that with the high-fat diet, there were some decreases in percent bone volume, bone surface, and trabecular number. A reduction in the total porosity can be seen in the exercise group. There was a slight increase in BMD with high-fat intervention in both trabecular and cortical bones. The method should be suitable to be applied to samples collected from in vivo experiments.
The number of BMAds was typically 300-500 /mm2 in the bone marrow region and the size range of cells was about 700 to 1000 μm2. Our optimized IHC method brings improvement over H&E as it can positively stain BMAds. It can be applied to histological bone tissue sections. An improved quantitation analysis based on this IHC method was presented by the end. Pilot studies from the μCT from different groups showed that with the high-fat diet, there were some decreases in percent bone volume, bone surface, and trabecular number. A reduction in the total porosity can be seen in the exercise group. There was a slight increase in BMD with high-fat intervention in both trabecular and cortical bones. The method should be suitable to be applied to samples collected from in vivo experiments.