Immunohistochemical Profiling of Cellular Markers

All IHC studies were performed on 5 µm FFPE sections using a Leica BOND RXm autostainer. Slides were stained with antibodies targeting human P-S6 (Cell Signaling #4858, 1:100) and P-PRAS40 (Cell Signaling #13175, 1:200) using a modified version of either the standard Leica Bond DAB “F” or red “J” IHC protocols. Slides were scored by a board-certified pathologist and given an H-score based on percentage and intensity of positivity. CD3, CD8, PAX5, and PTEN staining and analyses were performed as previously described.^{8 (link)}

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Fischer G.M., Guerrieri R.A., Hu Q., Joon A.Y., Kumar S., Haydu L.E., McQuade J.L., Vashisht Gopal Y.N., Knighton B., Deng W., Hudgens C.W., Lazar A.J., Tetzlaff M.T, & Davies M.A. (2021). Clinical, molecular, metabolic, and immune features associated with oxidative phosphorylation in melanoma brain metastases. Neuro-oncology Advances, 3(1), vdaa177.

Publication 2021

BOND RXm autostainer

Antibodies Human Pathologist Pax5 Pten

Corresponding Organization : The University of Texas MD Anderson Cancer Center

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Variable analysis

independent variables

Antibodies targeting human P-S6 (Cell Signaling #4858, 1:100) and P-PRAS40 (Cell Signaling #13175, 1:200)

dependent variables

Percentage and intensity of positivity for P-S6 and P-PRAS40 staining, scored by a board-certified pathologist using an H-score

control variables

5 μm FFPE sections
Leica BOND RXm autostainer
Modified version of either the standard Leica Bond DAB "F" or red "J" IHC protocols

positive controls

CD3, CD8, PAX5, and PTEN staining and analyses were performed as previously described

negative controls

Not explicitly mentioned

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