TMA sections were deparaffinized by using a Tissue-Tek® DRS™ instrument (Sakura). Endogenous peroxidase was blocked by immersion in 0,85% H2O2 (35% Arcos organics, the Netherlands) for 30 minutes at room temperature. ALK Antigen retrieval was performed by heating slides for 10 minutes in a target retrieval solution (EnVision™ FLEX Target Retrieval Solution HIGH pH, Denmark) and for p-ALK 15 min (EnVision™ FLEX Target Retrieval Solution LOW pH, Denmark) using a pressure cooker (decloaking chamber, Biocare Medical). ALK expression was detected using a rabbit monoclonal antibody (clone D5F3®, Cell Signaling Technology; dilution 1:150,) and the presence of p-ALK was detected using a monoclonal rabbit antibody (Tyr1604, Cell Signaling technology; dilution 1:25). Antibodies were diluted in a Normal antibody diluent (Normal antibody diluent, ImmunoLogic, the Netherlands). Sections were incubated with the primary antibodies overnight at +4°C. Antibodies were detected using BrightVision polyHRP-Anti-Rabbit IgG (ImmunoLogic, Amsterdam, The Netherlands) and immunostained using Vector laboratories ImmPACT[R] DAB Substrate Kit, Peroxidase (dilution 1 drop per 1 ml) for 5 minutes at room temperature. Finally, slides were counterstained using Mayers Hematoxylin (Lillie´s Modificatin, Dako, USA) and mounted (Eukitt® Quick-hardening mounting medium, SIGMA-ALDRICH®, Germany).
Clone d5f3
Manufactured by Cell Signaling Technology
Sourced in United States
Clone D5F3 is an antibody product offered by Cell Signaling Technology. It is a primary antibody that can be used for various research applications.
Automatically generated - may contain errors
Lab products found in correlation
Mayer s hematoxylin, by Agilent Technologies (1 mentions)
Eukitt quick hardening mounting medium, by Merck Group (1 mentions)
Decloaking chamber, by Biocare Medical (1 mentions)
Normal antibody diluent, by Immunologic (1 mentions)
Brightvision polyhrp anti rabbit igg, by Immunologic (1 mentions)
Clone d4d6, by Cell Signaling Technology (1 mentions)
Ros1 probe, by Abbott (1 mentions)
Bond 3, by Leica (1 mentions)
Real antibody diluent, by Agilent Technologies (1 mentions)
Clone d6b6, by Cell Signaling Technology (1 mentions)
Ventana benchmark xt, by Roche (1 mentions)
6 protocols using clone d5f3
1
Immunohistochemical Detection of ALK and p-ALK
2
Identification of ALK and ROS1 Rearrangements
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To identify ALK and ROS1 rearrangements, IHC was performed using the ALK (rabbit monoclonal, clone D5F3, Cell Signaling Technology, Danvers, MA, USA) and ROS1 (rabbit monoclonal, clone D4D6, Cell Signaling Technology, Danvers, MA, USA) antibodies. In IHC positive cases, fluorescence in situ hybridization (FISH) was performed using a break-apart ALK probe (Vysis LSI Dual Color, Break Apart Rearrangement Probe kit, Abbott Molecular, Abbott Park, IL, USA) or ROS1 probe (Abbott Molecular). ALK or ROS1 rearrangements were scored as positive when more than 15% of tumor cells displayed split signals or isolated signals containing a kinase domain (red for ALK and green for ROS1).
3
Immunohistochemical Antibody Staining
4
Immunohistochemical Analysis of ALK and EGFR Mutations
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TMAs and TBB samples, sectioned at a thickness of 4 m, were stained with a rabbit anti-ALK monoclonal primary antibody (Clone D5F3, Cell Signaling Technology, Danvers, MA, USA catalog number 3633, 1:500 dilution) in Dako REAL Antibody Diluent, using an automated IHC stainer (Leica Bond-III, Dako, Trappes, France). IHC results were scored based on the staining intensity and the proportion of stained cells. The staining-intensity categories were: 0 (no staining), 1 (mild staining), 2 (moderate staining), and 3 (heavy staining). The proportion of stained cells in each category was: 0 (no cells stained), 1 (1–20% of tumor cells stained), 2 (21–50% of tumor cells stained), and 3 (>51% of tumor cells stained). If both the intensity and proportion scores were 1 or more, the sample was defined as IHC-positive. Two common EGFR mutations (exon 19, codon E746–A750 deletion and exon 21, L858R point mutation) were examined in all TMA and TBB samples using rabbit EGFR mutation-specific monoclonal antibodies [clone D6B6 (catalog number 2085) and clone 43B2 (catalog number 3197), respectively; Cell Signaling Technology].
5
Immunohistochemical Detection of ALK
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Four micrometer sections cut from formalin-fixed, paraffin-embedded blocks were placed on charged slides, then dried and melted at 62°C for 20 min. Slides were placed on a Ventana BenchMark XT (Ventana Medical Systems Inc., Tucson, AZ) for staining. The staining protocol includes online deparaffinization, heat-induced epitope retrieval with Ventana Cell Conditioning 1 for 32 min, primary ALK antibody incubation for 32 min at 37°C (clone D5F3, a rabbit monoclonal antibody, 1:100 dilution, Cell Signaling Technology, Danvers, MA). Antigen–antibody reactions were visualized using Ventana Optiview Universal DAB Detection Kit. Counterstaining was carried out on the Ventana BenchMark XT using Ventana Hematoxylin II for 8 min, followed by bluing reagent for 4 min. The positive control was a known ALK-rearranged lung adenocarcinoma and the negative control was a mouse IgG1 serum substitution for the primary ALK antibody.
6
Detecting ALK and ROS1 Rearrangements
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To identify ALK and ROS1 rearrangements, IHC was performed using ALK (rabbit monoclonal, clone D5F3, Cell Signaling Technology, Danvers, MA) and ROS1 (rabbit monoclonal, clone D4D6, Cell Signaling Technology) antibodies, as previously described [7 (link)]. For IHC positive cases, FISH was performed using a break-apart ALK or ROS1 probe (Vysis LSI Dual Color, Break Apart Rearrangement Probe, Abbott Molecular, Abbot Park, IL), and ALK or ROS1 rearrangements were scored as positive when at least 15% of the tumor cells exhibited split or isolated 3′ signals.
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