Immunohistochemical Analysis of Tumor Markers

Tumors were processed for immunohistochemistry (IHC) as previously described (50 (link)). Sections were heated in 10mmol/L citrate buffer (pH 6.0) or 10mmol/L Tris+1mmol/L EDTA buffer (pH 9.0) with a decloaking chamber and incubated overnight at 4ºC with pS6rpY235/236 (1:200), Ki67 (1:800), or MERTK (1:50) antibodies. Bound antibodies were detected using the VECTASTAIN Universal Kit/HRP (Vector Laboratories) and 3,3’-diaminobenzidine substrates. Tissues were counterstained with Mayer hematoxylin (ThermoFisher Scientific) and were examined using an Olympus BX51 microscope. Images are shown at a magnification of 20X.

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McDaniel N.K., Cummings C.T., Iida M., Hülse J., Pearson H.E., Vasileiadi E., Parker R.E., Orbuch R.A., Ondracek O.J., Welke N.B., Kang G.H., Davies K.D., Wang X., Frye S.V., Earp H.S., Harari P.M., Kimple R.J., DeRyckere D., Graham D.K, & Wheeler D.L. (2018). MERTK mediates intrinsic and adaptive resistance to AXL-targeting agents. Molecular cancer therapeutics, 17(11), 2297-2308.

Publication 2018

VECTASTAIN Universal Kit/HRP Mayer hematoxylin BX51 microscope

Antibodies Buffer Citrate Edta Hematoxylin Immunohistochemistry Mertk Microscope Tissues Tris buffer Tumors Vector

Corresponding Organization : Emory University

Other organizations : University of Colorado Anschutz Medical Campus, University of North Carolina at Chapel Hill, University of North Carolina Health Care

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Variable analysis

independent variables

Heating method (10mmol/L citrate buffer (pH 6.0) or 10mmol/L Tris+1mmol/L EDTA buffer (pH 9.0) with a decloaking chamber)

dependent variables

Immunohistochemical staining and expression of pS6rpY235/236, Ki67, and MERTK

control variables

Incubation temperature (4ºC overnight)
Antibody dilutions (pS6rpY235/236 at 1:200, Ki67 at 1:800, MERTK at 1:50)
Detection method (VECTASTAIN Universal Kit/HRP and 3,3'-diaminobenzidine substrates)
Counterstaining (Mayer hematoxylin)
Microscopy (Olympus BX51 microscope, 20X magnification)

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

Annotations

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