EGFR and RAS gene mutations were analyzed in paraffin-embedded tissue sections from BM only. EGFR mutations (including G719X in exon 18, exon 19 deletion, T790M in exon 20, and L858R and L861Q in exon 21) and RAS mutations (G12C and G12A in exon 2 and Q61H in exon 3) were detected in matching formalin-fixed paraffin-embedded tissue samples using cycleave PCR (which was performed by SRL Inc., Tokyo, Japan, cobas® EGFR Mutation Test v2 kit and MEBGEN™ RASKET kit according to the manufacture's protocol). ALK rearrangement was analyzed by immunohistochemistry (ALK Detection kit; Nichirei Bioscience, Tokyo, Japan), according to the manufacturer's protocols. The slides were observed under ×200 magnification using a BX50 fluorescence microscope (Olympus Corporation, Tokyo, Japan).
Alk detection kit
Manufactured by Nichirei Biosciences
Sourced in Japan
The ALK Detection Kit is a laboratory tool used to detect the presence of the anaplastic lymphoma kinase (ALK) protein. It is designed to provide a reliable and efficient method for identifying ALK expression in various biological samples. The kit includes the necessary reagents and protocols to perform the ALK detection analysis.
Automatically generated - may contain errors
Lab products found in correlation
Bx50 fluorescence microscope, by Olympus (1 mentions)
Streptavidin horseradish peroxidase hrp, by Vector Laboratories (1 mentions)
Vysis alk break apart fish probe kit, by Abbott (1 mentions)
Oncomine dx target test, by Thermo Fisher Scientific (1 mentions)
Antigen retrieval solution, by Nichirei Biosciences (1 mentions)
6 protocols using alk detection kit
1
Molecular Profiling of Lung Cancers
2
Immunohistochemical Detection of EML4-ALK
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All Xuanwei samples were subjected to IHC to detect the EML4-ALK fusion protein. IHC was performed on 4-µm FFPE tissue sections placed on silane-coated slides using the 5A4 anti-ALK primary antibody in the ALK Detection kit (Nichirei Bioscience, Tokyo, Japan) (12 (link)). Tumor cells that stained more strongly in the cytoplasm compared with negative control cells, were defined as IHC-positive. Semi-quantitative assessment was performed by estimating the percentage of IHC-positive tumor cells. ALK IHC scores were assigned using the iAEP method (iScore) as follows: 0, no stained cells; 1, 0–50% stained tumor cells; 2, 50–80% stained tumor cells or >80% stained tumor cells with marked variability of staining intensity (‘checkerboard pattern’); and 3, >80% stained tumor cells without marked variability of staining intensity.
3
Immunohistochemical Evaluation of hNLRR1
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The six NB specimens were surgically collected and embedded in paraffin at Kyushu University Hospital. The appropriate informed consent was obtained under the institutional review board-approved protocol. The formalin-fixed, paraffin-embedded specimens and a purchased tissue array MC803a (U.S. Biomax) were deparaffinised and stained with an anti-hNLRR1 antibody (R&D, 1:250) overnight. The signal was detected by a biotin-conjugated secondary antibody and streptavidin-horseradish peroxidase (HRP) (VECTOR), followed by diaminobenzidine staining (VECTOR). An ALK detection kit (Nichirei) was used according to the manufacturer’s protocol. The study was approved by the Kyushu University of Medical Human Investigation Committee and the internal review board of the Chiba Cancer Center. The study was performed in accordance with the approved guideline.
4
Immunohistochemical Analysis of ALK
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Anti-ALK IHC was performed using the iAEP method (ALK Detection Kit, Nichirei Bioscience, Tokyo, Japan) with an automatic staining protocol13 (link),25 (link). To confirm ALK positivity, IHC (and/or FISH) was also performed and evaluated by a commercial clinical laboratory (SRL, Tokyo). ALK IHC results were classified using iScore for iAEP IHC14 (link),26 (link).
5
Post-alectinib treatment in ALK-rearranged NSCLC
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Patients with ALK-rearranged NSCLC who received LOR (LOR group [100 mg orally once daily]) or PEM (PEM group [500 mg/m2]: pemetrexed alone or combination with a platinum agent) as post-alectinib treatment between December 2012 and May 2020 at the National Cancer Center Hospital were included. The cutoff date for our analysis was March 30, 2021. Medical records, including patient characteristics and clinical outcomes, were retrospectively reviewed. The ALK gene rearrangement was identified by immunohistochemistry (ALK Detection Kit, Nichirei Bioscience, Tokyo, Japan; DF53, Roche, Basel, Switzerland; and 5A4, Abcam, Cambridge, United Kingdom), fluorescence in situ hybridization (Vysis ALK Break Apart FISH Probe Kit, Abbott Molecular, Abbott Park, IL), reverse-transcriptase polymerase chain reaction analysis, and next-generation sequencing (Oncomine Dx Target Test, Thermo Fisher Scientific, Waltham, MA).
6
Immunohistochemistry Analysis of ALK and P-gp
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Formalin-fixed, paraffin-embedded tissue specimens were sliced by a thickness of 4 μm, and the sections were placed on silane-coated slides. For antigen retrieval, slides were heated for 45 min at 105 °C in an antigen retrieval solution at pH 9.0 (Nichirei Biosciences, Inc., Tokyo, Japan). Antigen–antibody complexes were visualized with Histofine Simple Stain MAX PO detection reagent (Nichirei Biosciences, Inc.). The ALK Detection Kit (Nichirei Biosciences, Inc.), which is based on the intercalated antibody-enhanced polymer (iAEP) method (Takeuchi et al., 2009 (link)), was used for anti-ALK IHC analysis. The staining procedure was performed using the Histostainer automated staining system (Nichirei Biosciences, Inc.). For IHC of P-gp, anti-ABCB1 (P-gp) monoclonal antibody (6C4.2, Chemicon International Inc.) was used. Positivity for P-gp was standardized using KB3-1 (negative control) and ABCB1 overexpressed KB3-1 (KB3-1/ABCB1, positive control).
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