Immunohistochemical analysis of the formalin-fixed, paraffin-embedded (FFPE) tumor tissues for EGFR expression was performed using the Dako EGFR PharmDx TM kit (Dako, Glostrup, Denmark). The FFPE specimens were evaluated for both cytoplasmic and membranous immunostaining by two independent pathologists blinded to the clinical results (recorded at Fudan University Shanghai Cancer Center). The cases were divided into EGFR-positive and EGFR-negative subgroups based on the intensity and completeness of immunohistochemical staining. The staining intensity was scored as 0 for absent, 1 for weak, 2 for moderate and 3 for strong staining. The staining distribution was scored as 0 (absent) for <5%, 1 (focal) for 5% to <50%, and 2 (diffuse) for ≥50% positive-stained areas. The sum of intensity and distribution scores were then used to determine the EGFR immunoreactivity. For membranous or cytoplasmic staining, the intensity score of 1 and 0, or the distribution score of 0, was considered as negative immunoreactivity. Other scores were considered positive immunoreactivity.
Egfr pharmdx kit
Manufactured by Agilent Technologies
Sourced in Denmark, United States
The EGFR pharmDx kit is a qualitative immunohistochemical assay designed to detect the epidermal growth factor receptor (EGFR) protein in formalin-fixed, paraffin-embedded human non-small cell lung cancer (NSCLC) tissue.
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Lab products found in correlation
Herceptest, by Agilent Technologies (1 mentions)
Grp94, by Abcam (1 mentions)
Benchmark xt platform, by Roche (1 mentions)
Aperio scanscope cs slide scanner, by Leica (1 mentions)
Imagescope, by Leica (1 mentions)
Envision peroxidase detection system, by Agilent Technologies (1 mentions)
Chamber slide, by BD (1 mentions)
Envision flex kit, by Agilent Technologies (1 mentions)
Hsp70, by Abcam (1 mentions)
Ki 67p, by Dianova (1 mentions)
K5005, by Agilent Technologies (1 mentions)
Qiaamp dna ffpe tissue kit, by Qiagen (1 mentions)
16 protocols using egfr pharmdx kit
1
EGFR Expression Analysis in FFPE Tumor Tissues
2
Comprehensive ErbB Receptor Profiling in Pediatric Cancers
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EGFR and HER2 expression levels were determined using IHC assays previously validated in adult, but not pediatric, cancers (DAKO EGFR pharmDx TM Kit; Dako HercepTest). 38 Exploratory analysis of HER3/HER4 expression used IHC assays that are analytically validated for use in pediatric cancers.
Distribution of ErbB receptor expression was assessed based on staining intensity distribution, using the "magnification rule," 38 and calculation of a final Hirsch (H)-score, based on the following equation 39 :
Distribution of ErbB receptor expression was assessed based on staining intensity distribution, using the "magnification rule," 38 and calculation of a final Hirsch (H)-score, based on the following equation 39 :
3
Immunohistochemical Profiling of Tumor-Infiltrating Lymphocytes
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Immunohistochemical staining was performed on the four micrometer TMA sections. Immunostaining for CD3 (DAKO, Glostrup, Denmark), CD8 (Neomarkers, Fremont, CA, USA), and Foxp3 (Abcam, Cambridge, UK) were performed using a Leica BOND-MAX autostainer. For GRP94 (Abcam, Cambridge, UK) and CD4 (Ventana, Tucson, AZ, USA), immunohistochemical staining was performed using the BenchMark XT platform. For detection of EGFR mutations, we performed immunohistochemistry using the EGFR pharmDx kit (Dako, Glostrup, Denmark) following the manufacturer’s instructions. Moderate to strong immunostaining for GRP94 in more than 25% of tumor cells was defined as GRP94 positivity; EGFR-positivity was defined by the presence of any membranous immunostaining of tumor cells [34 (link)]. For quantitative TIL analysis, immunostained TMA slides were scanned using an Aperio ScanScope CS slide scanner (Aperio Technologies, Vista, CA, USA) at 40× magnification. After digitalization, the automated digital image analysis algorithm (ImageScope TM, Aperio Technologies, Vista, CA, USA) of Aperio was used to quantify CD3+, CD4+, CD8+, and Foxp3+ TILs, and the density of each TIL was recorded [35 (link)]. The density of each immunoreactive TIL was dichotomized into high and low groups based on the median values.
4
Immunohistochemical Evaluation of EGFR, ER, PR, and Ki-67 in FFPE Tissues
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Formalin-fixed and paraffin-embedded tissue sections were cut, dried, dewaxed in xylene and rehydrated through graded alcohol. Epidermal growth factor receptor expression was detected using EGFR pharmDx Kit (Dako, Capinteria, CA, USA) and scored as follows: 0, no staining, or weak membranous staining in <10% of the tumour cells; 1+, weak membranous staining in ⩾10% of the tumour cells; 2+, moderate, membranous staining in ⩾10% of the tumour cells; 3+, strong membranous staining in ⩾10% of the tumour cells. Both complete and incomplete membranous staining was accepted. If the triplicate TMA cores yielded different scores, the highest score was used. In the first set, we classified tumours into two groups according to the staining pattern of EGFR: homogeneous expression (0/1+ or 2+/3+ in all TMA cores) and heterogeneous expression (mixed pattern of 0/1+ and 2+/3+ in three TMA cores).
Oestrogen and progesterone receptors were regarded as positive if there were at least 1% positive tumour nuclei (Hammond et al, 2010 (link)). Cases with 10% or more positive staining were grouped as positive for p53. The Ki-67 proliferation index was defined as the percentage of tumour cells showing nuclear positivity.
Oestrogen and progesterone receptors were regarded as positive if there were at least 1% positive tumour nuclei (Hammond et al, 2010 (link)). Cases with 10% or more positive staining were grouped as positive for p53. The Ki-67 proliferation index was defined as the percentage of tumour cells showing nuclear positivity.
5
EGFR Expression and Phosphorylation Evaluation
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EGFR expression was detected by IHC using EGFR PharmDX kit (Dako, Glostrup, Denmark) according to the manufacturer's instructions. Antigen retrieval was performed treating sections by proteinase K. Phospho-EGF Receptor (Tyr1173) (EGFRtyr1173) and Phospho-EGF Receptor (Tyr1068) (EGFRtyr1068) expression was detected by IHC using specific monoclonal antibodies (Cell Signaling). Antigen retrieval was performed at 96 °C using a 10 mM EDTA buffer, pH 8, for 40 min in a thermostatic bath. Diaminobenzidine (DAB) was used as chromogenic substrate. EGFR, p-EGFRtyr1068 and p-EGFRtyr1173 were interpreted according to the follow scoring criteria: negative, no reaction; 1+, 2+ or 3+, if neoplastic cells displayed a weak, moderate or strong plasmamembrane immunostaining, respectively.
6
Immunohistochemical Analyses of Prostate Cancer
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The immunohistochemistry was performed using specific antibodies against mouse monoclonal Androgen Receptor (AR, clone 2F12, dilution 1:25, Novocastra, Dublin, OH, USA) [27 (link)], mouse monoclonal Cytokeratin 5/6 (CK5/6, Clone CK5/6.007, dilution 1:100, Biocare Medical, Concord, CA, USA) [28 (link)], mouse monoclonal p-AKT (Clone HP18, dilution 1:75, Novocastra), [29 (link)], rabbit monoclonal p-p44/42 MAPK (Clone 20G11, dilution 1:100, Cell Signaling Technology, Boston, MA, USA), [30 (link)] and mouse monoclonal PTEN (Clone 6M2.1, dilution 1:200, DakoCytomation, Glostrup, Denmark) [31 (link)]. Immunoreactions were obtained by incubating sections with specific primary antibodies for 15 minutes. Immunodetection was performed using a non-biotin highly sensitive system (EnVision Peroxidase Detection System, Dako), preventing possible false-positive staining due to endogenous biotin present in the tissue. The slides were then incubated with substrate chromogen solution diaminobenzidine (DAB) for 10 minutes and counterstained with haematoxylin. Specifically, mouse monoclonal EGFR (Clone 2-18C9) immunoreaction was executed using EGFR pharmDx™ Kit (DakoCytomation) [32 (link)], according to manufacturer’s instructions.
7
Pluripotency and Lineage Commitment Markers
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5 × 105 cells in WIT-P media were plated per well of a chamber slide (BD Falcon, San Jose, CA, USA) and the cells incubated overnight. Cells were fixed using 4% paraformadehyde, 0.15% picric acid in 1 × PBS for 20 minutes at RT. OCT4, NANOG and MyoD immunohistochemistry were performed per the protocols given in Additional file 1 : Table S2. EGFR IHC was performed using the EGFR (Dako) RTU primary antibody and the EGFR pharmDX Kit (Dako), which was used according to the manufacturer’s instructions.
8
Tumor EGFR Protein Expression Analysis
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Archived tumor tissue (pretreatment) derived from either the primary tumor or metastatic sites were collected and stored at a secure central laboratory. A tissue block or minimum of four tissue slides (paraffin embedded) was required for analyses. Tumor EGFR protein expression was assayed at a Clinical Laboratory Improvement Amendments (CLIA)-certified laboratory by IHC with the EGFR PharmDx Kit (Dako, Glostrup, Denmark) and evaluated independently by two trained pathologists to derive percent positive. Discordant results were jointly resolved by the two pathologists.
9
Immunohistochemical Analysis of EGFR, hMLH1, and hMSH2
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Immunohistochemical analysis was performed using the EGFR pharm Dx™ kit (DAKO, Glostrup, Denmark) and the EnVision™ FLEX kit (DAKO), according to the manufacturer’s instructions.
The sections were incubated with primary antibodies against hMLH1 (mouse anti-hMHL1: clone ES05 (DAKO), diluted 1:50, 30 min RT), hMSH2 (mouse anti-hMSH2: clone FE11 (Invitrogen), diluted 1:100, 20 min RT) and EGFR (mouse anti-EGFR: clone 2-18C9 (DAKO), diluted 1:100, 30 min RT).
The extent of staining was evaluated by visual examination microscopically. Nuclear staining (hMLH1 and hMSH2) was scored as “normal”, “low,” or “absent” compared with internal positive controls, according to previous scoring systems [12 (link), 13 (link)]. In the EGFR analysis, the scoring system recommended by Tsao et al. [24 (link)] was followed and results were recorded as positive (staining of ≥10% of membrane cells) or negative (staining of <10% of membrane cells).
The sections were incubated with primary antibodies against hMLH1 (mouse anti-hMHL1: clone ES05 (DAKO), diluted 1:50, 30 min RT), hMSH2 (mouse anti-hMSH2: clone FE11 (Invitrogen), diluted 1:100, 20 min RT) and EGFR (mouse anti-EGFR: clone 2-18C9 (DAKO), diluted 1:100, 30 min RT).
The extent of staining was evaluated by visual examination microscopically. Nuclear staining (hMLH1 and hMSH2) was scored as “normal”, “low,” or “absent” compared with internal positive controls, according to previous scoring systems [12 (link), 13 (link)]. In the EGFR analysis, the scoring system recommended by Tsao et al. [24 (link)] was followed and results were recorded as positive (staining of ≥10% of membrane cells) or negative (staining of <10% of membrane cells).
10
Immunohistochemical Analysis of Bax and Hsp70 Proteins in Wounded Tissue
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The Bax and Hsp70 protein expressions were evaluated in each formalin-fixed paraffin-embedded wounded tissue section, as previously described in detail.14 (link) Immunostaining was performed using the EGFR pharmDx™ kit (DakoC ytomation, Carpinteria, CA, USA), according to the manufacturer’s protocol. In brief, endogenous peroxidase activity was quenched using a peroxidase block. Tissue sections were then incubated with Bax (1:200, Cat: ab7977; Abcam, Cambridge, MA, USA) and Hsp70 (1:500, Cat: ab2787, Abcam) biotinylated primary antibody for 15 minutes followed by another 15 minutes of incubation with streptavidin–horseradish peroxidase. The sections were incubated with diaminobenzidine tetrahydrochloride for 5 minutes, and then counterstained with hematoxylin and 0.5% ammonia in water. The brown illustrations of samples under a light microscope demonstrated the positive findings.
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