Table 1

Studied proteins, primary antibodies, source and dilutions of antibodies, staining patterns, and interpretation (cut-off) criteria used in the present study
Protein Antibody [clone, source] Antibody dilution Time/Pretreatment Incubation Time Cutoff (%) Staining pattern
Cell adhesion molecules
  E-cadherin HECD-11 1:30 20 min / ER1 60 min IRS ≥5 C, C/M
  P-cadherin 562 1:200 20 min / ER2 O/N IRS ≥5 C, C/M
Cell assembly molecules
  Fascin-1 IM201 1:400 20 min / ER1 O/N 5% C
  Cytokeratin Multi AE1/AE31 1:100 10 min / Enzyme 1 20 min 1% C
Cell cycle regulators
  Cyclin D1 SP43 1:80 20 min / ER1 30 min IA (5%) N
Cell growth molecules
  VEGF-A VG14 1:75 20 min / ER2 60 min 5% C
  VEGF-C 18-2255, pl5 1:250 20 min / ER1 O/N 5% C
  Phospho-mTORSer2448 49 F96 1:30 20 min / ER1 20 min 5% C, C/PN
Cell proliferation molecules
  Ki67 MIB14 1:70 15 min / ER2 20 min IA (13%) N
Cell receptors
  EGFR 31 G75 1:50 8 min / Enzyme 2 20 min 10% M, C/M
  VEGFR-2 55B116 1:450 20 min / ER2 O/N 5% C, C/N
  VEGFR-3 KLT91 1:50 15 min / ER1 O/N 5% C
DNA repair molecules
  ERCC1 8 F12 1:300 15 min / ER2 30 min 50% N, C/N
  p53 DO-74 1:100 20 min / ER1 20 min 50% N, N/C
Inflammation mediators
  COX-2 4H121 1:300 20 min / ER1 O/N IRS≥3 C
Microtubule assembly
  MAPT (Tau) 2B2.1007 1:100 20 min / ER1 O/N 5% C, N, C/N
Molecules with kinase activity
  Phospho-AKTSer473 D9E6 1:150 20 min / ER2 O/N 5% C, N, C/N
  Phospho-GSK-3βSer9 5B36 1:50 20 min / ER1 O/N 5% C, N, C/N
  Phospho-p44/42 MAPKThr202/Tyr204 20 G116 1:1000 20 min/ ER1 O/N 5% C, N, C/N
Transcriptional activator or repressor
  p63 4A41 1:50 20 min / ER2 60 min 5% N
Tumor suppressor
  PTEN 6H2.14 1:300 20 min / ER2 60 min 10% C, N, C/N

1Leica Biosystems, Newcastle, UK; 2Thermo Fisher Scientific, Fremont, CA;3Spring Bioscience, Pleasanton, CA; 4Dako, Glostrup, Denmark; 5Invitrogen, Carlsbad, CA; 6CST: Cell Signaling Technology, Danvers, MA; 7United States Biological, Swampscott, MA.

ER1: Epitope Retrieval 1 (citric acid, pH 6); ER2: EDTA (pH 8.8); IA: image analysis; IRS: immunoreactive score=Intensity (0–3) +% of stained cells (0:0-5%, 1:6-25%, 2:26-50%, 3:>50%); min: minutes; M: membrane; N: nuclear; O/N: overnight; pl: polyclonal; PN: perinuclear; C: cytoplasm; C/M: cytoplasm/membrane.

Krikelis et al.

Krikelis et al. BMC Clinical Pathology 2013 13:1   doi:10.1186/1472-6890-13-1

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