Tissue Microarray Immunohistochemistry for pAKT and p27

TMAs were constructed in collaboration with the Unit of Pathology at INT Fondazione Pascale (Naples, Italy) according to established methods [69 ] using a Tissue Arrayer (Beecher Instruments, Gene Micro-Array Technologies, Silver Spring, MD). Immunostaining was performed using the avidin-biotin-peroxidase method (NovolinKT^M Polymer Detection System, Novacastra, Leica Biosystems) on formalin-fixed, paraffin-embedded tissues according to the manufacturer's protocol. Primary antibodies used were: anti-phosho-AKT (Ser473) (Cell Signalling Technology) used at dilution of 1:150 at pH 6, and anti-p27 (BD) used at dilution of 1:1000 at pH 6. The immunohistochemical score of pAKT used in this work was described previously [57 (link)]. As for p27, cut-off for scoring tumors as p27-positive or p27-negative was set at 50% [70 (link)]. p27 expression was scored nuclear in p27-positive tumors with exclusive nuclear staining and cytoplasmic in p27-positive tumors showing cytoplasmic staining independent of its nuclear positivity.

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De Marco C., Malanga D., Rinaldo N., De Vita F., Scrima M., Lovisa S., Fabris L., Carriero M.V., Franco R., Rizzuto A., Baldassarre G, & Viglietto G. (2015). Mutant AKT1-E17K is oncogenic in lung epithelial cells. Oncotarget, 6(37), 39634-39650.

Publication 2015

Tissue Arrayer anti-phosho-AKT (Ser473) anti-p27

Antibodies Avidin Biotin Cytoplasmic Dilution Embedded paraffin Formalin Gene Peroxidase Polymer Silver Tissue Tumors

Corresponding Organization :

Other organizations : Magna Graecia University, Biogem, Centre for Cancer Biology, Centro di Riferimento Oncologico, Istituto Nazionale Tumori IRCCS "Fondazione G. Pascale"

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Variable analysis

independent variables

Anti-phosho-AKT (Ser473) primary antibody (Cell Signalling Technology) used at dilution of 1:150 at pH 6
Anti-p27 primary antibody (BD) used at dilution of 1:1000 at pH 6

dependent variables

Immunohistochemical score of pAKT
P27 expression (scored as p27-positive or p27-negative based on 50% cut-off)

control variables

Formalin-fixed, paraffin-embedded tissues
Avidin-biotin-peroxidase method (NovolinKT^M Polymer Detection System, Novacastra, Leica Biosystems) used for immunostaining

controls

No positive or negative controls explicitly mentioned

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