Measuring Bone Marrow Adipocyte Density

To examine the effect of dietary hempseed on the density of bone marrow adipocytes, immunohistochemistry staining for the marker PRDM16 protein was employed. This zinc finger transcription factor plays an important role in the differentiation of adipocytes within the bone marrow [45 (link),46 (link),47 (link)]. Tissue was stained using a rabbit anti-human polyclonal antibody targeting PRDM16 (ThermoFisher Scientific, Waltham, MA, USA; catalog #PA5-20872). All other reagents were obtained from the Pierce Peroxidase Detection Kit (ThermoFisher Scientific; catalog #36000) and the protocol was followed according to the manufacturer’s instructions. Briefly, a goat anti-rabbit secondary antibody was applied to the tissues, followed by an anti-goat strep-HRP tertiary antibody. Samples were counterstained with hematoxylin, dehydrated and mounted. Slides were analyzed for PRDM16-positive cells (staining intensity) using a Leica DM6B widefield microscope and imaged with the attached Leica DFC450-C digital camera. Quantitative values were obtained by counting PRDM16-positive cells (brown stain) and total cells (blue stain) in the bone marrow and calculating a percentage.

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Blanton C.A., Barrott J.J., Kunz K., Bunde E., Streff H.M., Sparks C.A., Williams D.W, & Gabaldόn A.M. (2022). The Impact of Hempseed Consumption on Bone Parameters and Body Composition in Growing Female C57BL/6 Mice. International Journal of Environmental Research and Public Health, 19(10), 5839.

Publication 2022

Pierce Peroxidase Detection Kit DM6B widefield microscope DFC450-C digital camera

Adipocytes Anti antibody Bone density Bone marrow Cells Dietary Goat Hematoxylin Human Marrow Microscope Peroxidase Prdm16 Protein Rabbit Stain Strep Tissues Transcription factor Zinc finger

Corresponding Organization : Idaho State University

Other organizations : Colorado State University Pueblo, Harvard University

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Variable analysis

independent variables

Dietary hempseed

dependent variables

Density of bone marrow adipocytes

control variables

Procedure for immunohistochemistry staining using PRDM16 antibody
Reagents from Pierce Peroxidase Detection Kit
Hematoxylin counterstain
Microscopy and imaging equipment

controls

Positive control: Not specified
Negative control: Not specified

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