Appendix1: Details of IHC

Appendix1:

Supporting InformationMethodological details for immunohistochemistry, with all antigens tested on a tissue microarray from the cohort of 131 non-mucinous carcinomas. Antigen, supplier, catalogues, clone, method for scoring, concentration(s), methodological details, score, and cut-offs are outlined. Location of target antigen within tumor cells (N=nuclear, C=cytoplasm, M=membrane) or adjacent lymphocytes (L) is provided.

Antigen / Supplier / Catalogue / Clone / Method for scoring / Concen-tration / Details / Score / % / Target
ANXA4 / Abnova / H00000307-M13 / mouse clone 1D3 / Manual / 1:25 / 30min Citrate pH6.0, 30min Dako Serum Free Protein Block, primary overnight at 4C, secondary Dako EnVision 30min / 0
1 / None
Any / C
BRCA1 / Calbiochem / OP92 / mouse clone MS110 / Ventana Discovery XT
and Manual / 1:10/1:25/
1:50/1:75 / Std CC2, primary 2hr without heat, UltraMap kit / 0
1 / 1%
>1% / N
CyclinE / Signet / 903-01 / mouse clone HE12 / Ventana Discovery XT / 1:20 / Mild CC1, primary 60min with heat, DABMap kit / 0
1
2 / 5%
>5%
>80% / N
MDM2 / Neomarkers / MS-291 / mouse clone SMP14 / Ventana Discovery XT / 1:300 / Std CC1, primary 60min without heat, UltraMap kit / 0
1 / None
Any / N
NDRG1 / Abnova / H00010397-M03 / mouse clone 2D7 / Manual / 1:25 / 30min Citrate pH6.0, 30min Dako Serum Free Protein Block, primary overnight at 4C, secondary Dako EnVision 30min / 1
2 / Weak
Strong / C
p16 / mtm Laboratories / 9518 / mouse clone E6H4 / Ventana Discovery XT / 1:2 / Std CC1, primary 2hr without heat, UItraMap kit / 0
1
2
(and 0 vs. 1) / 5%
>5 %
>80% / N/C
p21 / Neomarkers / MS-230 / mouse clone DCS-60.2 / Ventana Discovery XT / 1:40 / Mild CC1, primary 32min with heat, DABMap kit / 0
1 / 5%
>5% / N
p27 / BD Transduction / 610241 / mouse clone 57 / Ventana Discovery XT / 1:50 / Mild CC2, primary 32min with heat, DABMap kit / 0
1
2 / 5%
>5-50
>50% / N
p53 / Dako / M7001 / mouse clone DO-7 / Ventana Discovery XT / 1:400 / Std CC1, primary 60min with heat, DABMap kit / 0
1
2 / Neg
1-80
>80% / N
pAKT / Cell Signaling / 3787 / rabbit clone 736E11 / Ventana Discovery XT / 1:50 / Std CC1, primary 2hr without heat, UItraMap kit / 0
1
2 / Neg
Weak
Strong / C
SERBP1 / Abnova / H00026135-M02 / mouse clone 1G5-2D7 / Manual / 1:50 / 30min Citrate pH6.0, 30min Dako Serum Free Protein Block, primary overnight at 4C, secondary Dako EnVision 30min / 1
2 / Weak
Strong / N/C
PTEN / Cell Signaling / 9559 / rabbit clone 138G6 / Ventana Discovery XT / 1:25 / Std CC1, primary 60min without heat, UltraMap kit / 0
1 / None
Any / N/C
CD8 / Dako / M7103 / mouse clone C8/144B / Ventana BenchMark XT / 1:100 / Mild CC1, primary 32min with heat, iView kit / 0
1 / None
Any / L
CD3 / Lab Vision / RM-9107 / rabbit clone SP7 / Ventana Discovery XT / 1:150 / Std CC1, primary 60min, DABMap kit / 0
1 / None
Any / L
CD4 / Lab Vision / MS-1528 / mouse clone 4B12 / Ventana Discovery XT / 1:100, 1:10 / Std CC1, primary 60min, DABMap kit / 0
1 / None
Any / L
CD20 / Lab Vision / RB-9013 / rabbit polyclonal / Ventana Discovery XT / 1:400 / Std CC1, primary 60min, DABMap kit / 0
1 / None
Any / L
FOXP3 / eBioscience / 14-7979 / mouse clone eBio7979 / Ventana Discovery XT / 1:50 / Std CC1, primary 60min, DABMap kit / 0
1 / None
Any / L
TIA1 / Abcam / ab2712 / mouse clone TIA-1 / Ventana Discovery XT / 1:50 / Std CC1, primary 60min, DABMap kit / 0
1 / None
Any / L

Appendix 1. Antigen, supplier, catalogues, clone, method for scoring, concentration(s), methodological details, score, and cut-offs are outlined. Location of target antigen within tumor cells (N=nuclear, C=cytoplasm, M=membrane) or adjacent lymphocytes (L) is provided.