IHC Protocol for Protein Expression

IHC for TMAs was performed as described previously [18 (link)]. Serial sections (approximately 5 μm) were stained with antibodies as previously described [5 (link)]. Briefly, sections were submitted to antigen retrieval (CC1, Ventana Medical Systems, Tucson, AZ, USA) using an auto-immunostainer (Discovery Staining Module, Ventana Medical Systems), followed by one-hour incubation with primary antibody and 32-minute incubation with secondary antibody. Primary antibody concentrations initially applied to the Ventana instrument were 1:50 for antibodies to p-S2448 mTOR, mTOR, p70S6K and p-T389 p70S6K translating into final concentrations of 1:150 after 1:3 dilution with buffer dispensed onto the slide with the primary antibody.

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Shrivastav A., Bruce M.C., Jaksic D., Bader T., Seekallu S., Penner C., Nugent Z., Watson P, & Murphy L. (2014). The mechanistic target for rapamycin pathway is related to the phosphorylation score for estrogen receptor-α in human breast tumors in vivo. Breast Cancer Research : BCR, 16(3), R49.

Publication 2014

Discovery Staining Module

Antibodies Antibody Antigen Buffer Dilution Mtor P70s6k

Corresponding Organization :

Other organizations : CancerCare Manitoba, University of Manitoba, University of Winnipeg

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Variable analysis

independent variables

Antibodies to p-S2448 mTOR, mTOR, p70S6K and p-T389 p70S6K

dependent variables

Expression levels of p-S2448 mTOR, mTOR, p70S6K and p-T389 p70S6K

control variables

Antigen retrieval (CC1, Ventana Medical Systems, Tucson, AZ, USA) using an auto-immunostainer (Discovery Staining Module, Ventana Medical Systems)
One-hour incubation with primary antibody and 32-minute incubation with secondary antibody
Primary antibody concentrations initially applied to the Ventana instrument were 1:50, translating into final concentrations of 1:150 after 1:3 dilution with buffer dispensed onto the slide with the primary antibody

controls

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

Annotations

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