Immunohistochemistry (IHC) was performed using the Ptenlox5/lox5;SSM2/SSM2 metastatic mouse model. Tissue was stained using a rabbit anti-human/mouse polyclonal antibody targeting either Oncostatin M Receptor (OSMR) (ABclonal, Woburn, MA, USA. Cat.# A6681) or eGFP (Thermo Fisher, Carlsbad, CA, USA. Cat.# CAB4211). All other reagents were obtained from the Pierce Peroxidase Detection Kit (Thermofisher, Carlsbad, CA, USA. Cat.# 36000) and protocol was followed according to the manufacturer’s protocol. 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 through a Leica DM6B widefield microscope and imaged with the attached Leica DFC450-C digital camera.
Dm6b widefield microscope
Manufactured by Leica
The DM6B widefield microscope is a high-performance optical instrument designed for a variety of scientific and research applications. It features a stable and ergonomic design, advanced optics, and a user-friendly interface. The DM6B provides reliable and consistent imaging capabilities for a wide range of samples.
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3 protocols using dm6b widefield microscope
1
Immunohistochemistry of Metastatic Tumor Model
2
Optimized U-ExM Protocol for Centriole Expansion
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We optimized our previously described U-ExM protocol (16 ) to obtain better expansion of centrioles within human cells. Two steps were modified as follows: the solution of FA/AA [FA 0.7 and 1% AA = 1X (16); FA 1.4 and 2% AA = 2X; and FA 2.1 and 3% AA = 3X) and the denaturation time (30, 60, and 90 min). The results of the optimization are presented in fig. S8 (A and B). The 2X protocol combined with 90-min denaturation at 95°C yielded the best expansion and was used for further analysis.
Confocal microscopy was performed on a Leica TCS SP8 using a 63× 1.4 numerical aperture (NA) oil objective, with the lightning mode at max resolution to generate deconvolved images, with the following parameters: Lightning at max resolution, adaptive as “Strategy” and water as “Mounting Medium.” 3D z stacks were acquired with 0.12-μm z intervals and an x,y pixel size of 35 nm. For fig. S8 (A and B), centrioles were imaged with a 100× 1.25 NA N PLAN oil objective on a Leica DM6B wide-field microscope, equipped with a scientific complementary metal-oxide semiconductor (sCMOS) monochrome Leica DFC9000 camera. Deconvolved images were generated with the Thunder “Small volume computational clearing” mode.
Confocal microscopy was performed on a Leica TCS SP8 using a 63× 1.4 numerical aperture (NA) oil objective, with the lightning mode at max resolution to generate deconvolved images, with the following parameters: Lightning at max resolution, adaptive as “Strategy” and water as “Mounting Medium.” 3D z stacks were acquired with 0.12-μm z intervals and an x,y pixel size of 35 nm. For fig. S8 (A and B), centrioles were imaged with a 100× 1.25 NA N PLAN oil objective on a Leica DM6B wide-field microscope, equipped with a scientific complementary metal-oxide semiconductor (sCMOS) monochrome Leica DFC9000 camera. Deconvolved images were generated with the Thunder “Small volume computational clearing” mode.
3
Measuring Bone Marrow Adipocyte Density
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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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