Immunostaining of Mouse Brain and 3D Spheroids

Mouse brains were fixed with 4% paraformaldehyde, 70μm sections were cut by cryostat (Thermal Fisher) and whole mount staining was applied following established protocols (12 (link)). For immunostaining of 3D cultured cell spheroids, cells were fixed with 4% paraformaldehyde and stained. For clinical samples, paraffin sections were stained by Wistar Histotechnology Facility. Antibodies used for immunochemical staining are listed in Supplementary Fig. 8. Images were acquired with Nikon 80i microscope (Nikon Instrument) or TCS SP5II upright confocal microscope (Leica), and analyzed with LAS AF and NIS-Elements software.

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Zou Y., Watters A., Cheng N., Perry C.E., Xu K., Alicea G.M., Parris J.L., Baraban E., Ray P., Nayak A., Xu X., Herlyn M., Murphy M.E., Weeraratna A.T., Schug Z.T, & Chen Q. (2019). Polyunsaturated Fatty Acids from Astrocytes Activate PPAR Gamma Signaling in Cancer Cells to Promote Brain Metastasis. Cancer discovery, 9(12), 1720-1735.

Publication 2019

Nikon 80i microscope TCS SP5II upright confocal microscope

Antibodies Brains Cells Confocal microscope Microscope Mouse Paraffin Paraformaldehyde

Corresponding Organization : The Wistar Institute

Other organizations : Boston University, University of Pennsylvania, Christiana Care Health System

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Variable analysis

independent variables

Mouse brain tissue fixation with 4% paraformaldehyde
Cutting 70μm sections by cryostat
3D cell culture spheroid fixation with 4% paraformaldehyde

dependent variables

Whole mount staining of mouse brain sections
Immunostaining of 3D cell culture spheroids
Immunochemical staining of clinical paraffin sections

control variables

Established protocols for whole mount staining (12)
Wistar Histotechnology Facility for clinical sample staining

positive controls

Not explicitly mentioned

negative controls

Not explicitly mentioned

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