Polymer refine detection kit

Manufactured by Leica

Sourced in Germany

The Polymer Refine Detection Kit is a laboratory equipment product designed for use in scientific research and analysis. It serves as a detection tool, providing a means to identify and analyze specific polymers. The kit's core function is to enable the identification and characterization of polymers through the application of specialized techniques and reagents. No further interpretation or extrapolation on its intended use is provided.

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Lab products found in correlation

Bond max, by Leica (6 mentions) 3 3 diaminobenzidine (dab), by Leica (3 mentions) Bond rxm system, by Leica (2 mentions) Bond rx autostainer, by Leica (2 mentions) Bond 3, by Leica (2 mentions) Protein block, by Agilent Technologies (1 mentions) Background reducing diluent, by Agilent Technologies (1 mentions) Hematoxylin, by Leica (1 mentions) Bond rx, by Leica (1 mentions) Osteosoft, by Merck Group (1 mentions) Axiovision software version 4, by Zeiss (1 mentions) Axioplan 2 microscope, by Zeiss (1 mentions) Rbm961l, by Biocare Medical (1 mentions) Pan akt, by Cell Signaling Technology (1 mentions) P egfr, by Cell Signaling Technology (1 mentions) Pfrs2 y436, by Abcam (1 mentions) P akt, by Cell Signaling Technology (1 mentions) Rodent block m, by Biocare Medical (1 mentions) Dako x0909, by Agilent Technologies (1 mentions) Bond polymer refine detection kit, by Leica (1 mentions)

22 protocols using polymer refine detection kit

Immunohistochemical Analysis of Tumor Markers

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Tissue sectioning and IHC staining with S100A9, CD68, and CD14 were performed at the Pathology Research Core (Mayo Clinic, Rochester, MN) using the Leica Bond RX stainer (Leica). Formalin-fixed, paraffin-embedded (FFPE) tissue from the primary tumor and corresponding SLN were identified for Patients 1 and 2, sectioned at 5 μm and IHC staining was performed. Slides for S100A9 stain were retrieved for 20 min using Epitope Retrieval 1 (Citrate; Leica) and incubated in Protein Block (Dako) for 5 min. The primary antibodies S100A9 (Sigma, HPA004193), CD68 (Dako, PG-M1 clone), and CD14 (Sigma, HPA001887) were diluted to 1:3000, 1:200, and 1:200 in Background Reducing Diluent (Dako) respectively, and incubated for 15 min. The detection system used was a Polymer Refine Detection Kit (Leica). Slides were counterstained with hematoxylin for 5 min followed by serial washes in 1X Bond wash buffer, distilled water and tap water. Slides were dehydrated in increasing concentrations of ethyl alcohol and cleared in three changes of xylene prior to permanent coverslipping in xylene-based medium. The immunostaining results were assessed by an expert, clinical pathologist to determine localization patterns in each case.

Maus R.L., Jakub J.W., Hieken T.J., Nevala W.K., Christensen T.A., Sutor S.L., Flotte T.J, & Markovic S.N. (2019). Identification of novel, immune-mediating extracellular vesicles in human lymphatic effluent draining primary cutaneous melanoma. Oncoimmunology, 8(12), e1667742.

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Immunohistochemical Profiling of ATM, pRAD50, and SLFN11

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All IHC used 4- to 5-μm-thick sections of formalin-fixed paraffin-embedded clinical tissues. ATM IHC was performed as described in Villaruz and colleagues (14 (link)). pRAD50 IHC was performed as described in Jones and colleagues (9 (link)). SLFN11 IHC was performed using a Leica Bond RX staining platform, the steps of which included dewaxing, pH9 antigen retrieval (ER2) for 25 minutes, protein blocking with SignalStain (Cell Signaling Technology), staining with 2.5 μg/mL anti-SLFN11 ab121731 antibody (Abcam), detection using the Polymer Refine Detection Kit (Leica), and counterstaining with hematoxylin (Leica). Appropriate control tissues and matched isotype controls were used during each staining run. Slides were digitalized using an Aperio AT2 scanner and pathology H-scores generated. H-scores are a product of the intensity of expression (0–3+) and percentage of cells stained. For ATM and SLFN11, which are both expressed in the nucleus, internal tissue control staining (e.g., lymphocytes) must be of 2+ or higher intensity for the sample to be deemed evaluable. A single pathologist performed the scoring at 20× magnification and the entire sample was assessed.

Yap T.A., Krebs M.G., Postel-Vinay S., El-Khouiery A., Soria J.C., Lopez J., Berges A., Cheung S.Y., Irurzun-Arana I., Goldwin A., Felicetti B., Jones G.N., Lau A., Frewer P., Pierce A.J., Clack G., Stephens C., Smith S.A., Dean E, & Hollingsworth S.J. (2021). Ceralasertib (AZD6738), an Oral ATR Kinase Inhibitor, in Combination with Carboplatin in Patients with Advanced Solid Tumors: A Phase I Study. Clinical Cancer Research, 27(19), 5213-5224.

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Histological analysis of mouse tissues

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Mice were euthanized under deep anaesthesia by intracardial perfusion with PBS followed by perfusion with 4% (w/v) paraformaldehyde (PFA) dissolved in PBS. All of the organs were removed and fixed in 4% PFA overnight. Vertebral columns, including the spinal cords, were additionally decalcified with Osteosoft (Sigma-Aldrich) for 72 h before paraffin embedding; 2-μm-thick sections were prepared. Immunohistochemistry was performed using the Leica Bond Rxm System with the Polymer Refine detection kit (Leica). A list of all of the antibodies used is provided in Supplementary Table 2. DAB was used as chromogen, and counterstaining was performed with haematoxylin. The slides were then scanned on the Leica AT2 system, and the images were analysed using QuPath v.0.3.2 (https://qupath.github.io, University of Edinburgh, Scotland).
Quantification of histological samples was performed automatically with computer-assisted algorithms provided by QuPath. To detect the total cell counts, regions of interest were annotated and analysed automatically by positive nuclear detection. All annotations were performed in a blinded manner.

Afzali A.M., Nirschl L., Sie C., Pfaller M., Ulianov O., Hassler T., Federle C., Petrozziello E., Kalluri S.R., Chen H.H., Tyystjärvi S., Muschaweckh A., Lammens K., Delbridge C., Büttner A., Steiger K., Seyhan G., Ottersen O.P., Öllinger R., Rad R., Jarosch S., Straub A., Mühlbauer A., Grassmann S., Hemmer B., Böttcher J.P., Wagner I., Kreutzfeldt M., Merkler D., Pardàs I.B., Schmidt Supprian M., Buchholz V.R., Heink S., Busch D.H., Klein L, & Korn T. (2024). B cells orchestrate tolerance to the neuromyelitis optica autoantigen AQP4. Nature, 627(8003), 407-415.

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Phospho-Aurora A Immunohistochemistry of Tumor Xenografts

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Formalin fixed, paraffin embedded tumor xenografts were sectioned at 5 μm and stained for anti-Phospho-Aurora A (phospho T288) protein. Antigen was retrieved on-line with the Leica Bond RX stainer (Leica, Buffalo, IL) using Epitope Retrieval Solution 1 (Citrate based solution, pH 6). The tissues were incubated in Rodent Block M by Biocare RBM961L for 30min. Primary anti-Phospho-Aurora A antibody was diluted in Dako background reducing diluent and incubated for 30min. Staining was conducted using polymer refine detection kit from Leica that includes DAB per manufacturer’s recommendation. Primary anti-Phospho-Aurora A (phospho T288) antibody (Abcam, ab58494) was used at 1:300 dilutions. A Zeiss Axioplan 2 microscope and Axiovision software (version 4.7) were used to generate images under bright field configured with 20x PlanNEOFLUAR (NA 0.50) and 40x N-ACHROPLAN (NA 0.65).

Kurokawa C., Geekiyanage H., Allen C., Iankov I., Schroeder M., Carlson B., Bakken K., Sarkaria J., Ecsedy J., D’Assoro A., Friday B, & Galanis E. (2016). Alisertib demonstrates significant antitumor activity in bevacizumab resistant, patient derived orthotopic models of glioblastoma. Journal of neuro-oncology, 131(1), 41-48.

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Immunohistochemistry for FGFR2, FGFR3, and AKT Signaling

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The immunohistochemistry was performed following the procedures described previously [110] (link). Briefly, slides were dewaxed, rehydrated and antigen retrieved on-line on the BondMax autostainer (Leica Microsystems, INC Bannockburn, IL). Slides were then subjected to heat-induced epitope retrieval using a proprietary EDTA-based retrieval solution. Endogenous peroxidase was then blocked and slides were incubated with the following antibodies: FGFR2 (BEK, Santa Cruz, catalog# sc-20735), FGFR3 (C-15, Santa Cruz, catalog# sc-123), panAKT (Cell Signaling Technology, catalog# 4685, pAKT (Cell Signaling Technology, catalog# 4060), EGFR (Cell Signaling Technology, catalog# 4267, pEGFR (Cell Signaling Technology, catalog#2234), MAPK/ERK1/2 (Cell Signaling Technology, catalog# 4695), pMAPK/pERK (Cell Signaling Technology, catalog# 4376) and pFRS2 Y436 (Abcam, catalog# ab78195). Sections were visualized using the Polymer Refine Detection kit (Leica) using diaminobenzidine chromogen as substrate.

Borad M.J., Champion M.D., Egan J.B., Liang W.S., Fonseca R., Bryce A.H., McCullough A.E., Barrett M.T., Hunt K., Patel M.D., Young S.W., Collins J.M., Silva A.C., Condjella R.M., Block M., McWilliams R.R., Lazaridis K.N., Klee E.W., Bible K.C., Harris P., Oliver G.R., Bhavsar J.D., Nair A.A., Middha S., Asmann Y., Kocher J.P., Schahl K., Kipp B.R., Barr Fritcher E.G., Baker A., Aldrich J., Kurdoglu A., Izatt T., Christoforides A., Cherni I., Nasser S., Reiman R., Phillips L., McDonald J., Adkins J., Mastrian S.D., Placek P., Watanabe A.T., LoBello J., Han H., Von Hoff D., Craig D.W., Stewart A.K, & Carpten J.D. (2014). Integrated Genomic Characterization Reveals Novel, Therapeutically Relevant Drug Targets in FGFR and EGFR Pathways in Sporadic Intrahepatic Cholangiocarcinoma. PLoS Genetics, 10(2), e1004135.

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Immunohistochemical Analysis of PVRL4 and MV Proteins

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Frozen human tissues and formalin fixed xenograft tissues were sectioned at 5 μm. Antigen retrieval and staining was conducted using Bond III biosystem, (PVRL4-citrate, PH 6; MV-F-EDTA, pH 9) and polymer refine detection kit (Leica) according to manufacturer’s recommendation. Primary antibodies were diluted in Dako background reducing diluent and incubated for 30min. The frozen tissue tissue samples were incubated in Dako-X0909, while xenograft tissues were incubated in Rodent Block-M (Biocare). PVRL4 primary antibody (Sigma, HPA010775) was used on brain (1:50), breast (1:100) and ovarian (1:200) tissue samples. MV fusion protein primary antibody (Abbiotec, 251363) was used on xenograft tissues at 1:100 dilutions. An Axioplan2 microscope was used with AxioCam HR camera and Axiovision software (version 4.7) to generate images under bright field, configured with 20x PlanNEOFLUAR (NA 0.50) and 40x N-ACHROPLAN (NA 0.65).

Geekiyanage H, & Galanis E. (2016). MiR‐31 and miR‐128 regulates poliovirus receptor‐related 4 mediated measles virus infectivity in tumors. Molecular Oncology, 10(9), 1387-1403.

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Quantifying Hepatocyte HBcAg Expression

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Sections (2 μm) of 4% paraformaldehyde-fixed, paraffin-embedded liver samples were stained using a BondMAX immunohistochemistry robot (Leica) using Bond solutions and Polymer Refine Detection Kit (Leica). Primary antibody HBcAg (RB1413A, StartFragmentLab VisionEndFragment) was used at a 1:50 dilution. Primary antibody was detected with Bond Polymer Refine Detection Kit from Leica (rabbit, Leica, DS9800).
For quantification of HBcAg staining, whole slides were scanned at ×40 magnification using a SCN400 slide scanner (Leica) and analyzed withTissue IA image analysis software (Slidepath, Leica) using an optimized color detection setting for DAB (positive) and Hematoxilin (negative) nuclei at ×20 magnification. For quantification, 3 random areas of 5–10 mm² in size on each slide were chosen and obtained values were merged for further statistical analysis. For HBcAg-positive hepatocytes, data are presented as positive nuclei (DAB positive nuclei) as a percentage of total nuclei in liver (DAB/Hematoxylin+, including hepatocytes and non-parenchymal cells).

Burwitz B.J., Wettengel J.M., Mück-Häusl M.A., Ringelhan M., Ko C., Festag M.M., Hammond K.B., Northrup M., Bimber B.N., Jacob T., Reed J.S., Norris R., Park B., Moller-Tank S., Esser K., Greene J.M., Wu H.L., Abdulhaqq S., Webb G., Sutton W.F., Klug A., Swanson T., Legasse A.W., Vu T.Q., Asokan A., Haigwood N.L., Protzer U, & Sacha J.B. (2017). Hepatocytic expression of human sodium-taurocholate cotransporting polypeptide enables hepatitis B virus infection of macaques. Nature Communications, 8, 2146.

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Kidney Histopathology and Apoptosis Quantification

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Kidneys were fixed in 4% formalin in PBS and embedded in paraffin. Sections of 2 μm were cut. Renal lesions were scored on sections stained with Hematoxylin-Eosin and PAS according to standard protocols. Cleaved Caspase 3 immunohistochemistry was performed on a BondRxm system (Leica, Wetzlar, Germany). Briefly, the slides were deparaffinized, and heat-mediated retrieval (ER1) was applied for 20 min. A primary antibody against cleaved Caspase 3 was used (1:150, Cell Signaling, Danvers, cat. No. 9664), and binding was visualized using a Polymer Refine Detection Kit (Leica, Wetzlar, Germany) without postprimary antibody. All slides were scanned with an AT2 scanning system (Leica, Wetzlar, Germany).
Renal tubular injury was scored by the percentage of injured tubules with loss of brush border, cell lysis, and cast formation: 0, no damage; 1, <25%; 2, 25 to 50%; 3, 50 to 75%; 4, >75% (60 (link)). Cleaved Caspase 3 positive tubular epithelial cells were counted in 10 high power fields (40×) and averaged.

Salei N., Ji X., Pakalniškytė D., Kuentzel V., Rambichler S., Li N., Moser M., Steiger K., Buch T., Anders H.J, & Schraml B.U. (2021). Selective depletion of a CD64-expressing phagocyte subset mediates protection against toxic kidney injury and failure. Proceedings of the National Academy of Sciences of the United States of America, 118(39), e2022311118.

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Ferritin Immunofluorescence Detection Protocol

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The immunofluorescence (IF) detection of ferritin was performed at the Molecular Cytology Core Facility of Memorial Sloan Kettering Cancer Center. Automated IF staining was conducted using Leica Bond BX staining system. Cells cultured on chamber slides were fixed in 4 % paraformaldehyde (PFA, Electron Microscopy Sciences, 157–8–100) for 15 min followed by washing in PBS. Slides were loaded in Leica Bond and IF staining was performed as follows. The primary antibodies against FTH1 (Rb, 0.11μg/mL, Cell signaling technology, 4393) were incubated with the sample for 1 hour at room temperature. After that, the Leica Bond Polymer anti-rabbit HRP secondary antibody (included in Polymer Refine Detection Kit (Leica, DS9800)) was used for 8 min, followed by incubation with Alexa Fluor tyramide signal amplification reagents (Life Technologies, B40953) for 10 min. After the run was finished, slides were washed in PBS and incubated in 5 μg/mL 4’,6-diamidino-2-phenylindole (DAPI) (Sigma Aldrich, D9542) in PBS for 5 min, rinsed in PBS, and mounted in Mowiol 4–88 (Calbiochem). Slides were kept overnight at - 20°C before imaging. The slides were scanned with a 20x/0.8NA objective on a Pannoramic Confocal Scanner (3DHistech, Budapest, Hungary).

Richard N., Salomon H., Rando R., Mansour T., Bowlin T.L, & Wainberg M.A. (2000). Selection and Characterization of Human Immunodeficiency Virus Type 1 Variants Resistant to the (+) and (−) Enantiomers of 2′-Deoxy-3′-Oxa-4′-Thio-5-Fluorocytidine. Antimicrobial Agents and Chemotherapy, 44(5), 1127-1131.

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Profiling Tumor Progression in MET-driven Lung Cancer

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Patients receiving targeted therapies at Massachusetts General Hospital undergo repeat biopsy at time of progression and are enrolled in an institutional review board-approved protocol of tissue collection and research. A patient who was receiving crizotinib therapy for metastatic lung cancer with MET exon 14 skipping underwent a biopsy at time of progression. Tumor genotyping on the baseline and postprogression biopsy samples was performed with the Snapshot nextgeneration sequencing version 1 assay, which uses anchored multiplex polymerase chain reaction for the detection of single nucleotide variants and insertion/ deletions in the genomic DNA of 39 cancer-related genes. 10 Immunohistochemical testing was performed on 5-mm sections of formalin-fixed paraffin-embedded tumor tissue. Total MET staining was performed using the CONFIRM Anti-Total c-MET (SP44) rabbit monoclonal primary antibody (Ventana Medical Systems, Tucson, AZ), and phospho-MET staining was performed using the Anti-MET phospho Y1349 rabbit monoclonal primary antibody (EP2367Y; ab68141) (Abcam, Cambridge, MA) diluted 1:100 in Leica Primary Antibody Diluent (Leica Biosystems Richmond, Inc., Richmond, IL). Targets were retrieved using Epitope Retrieval Solution 2 for 20 minutes, and staining was performed on the Leica BOND RX autostainer using the Polymer-Refine Detection Kit.

Heist R.S., Sequist L.V., Borger D., Gainor J.F., Arellano R.S., Le L.P., Dias-Santagata D., Clark J.W., Engelman J.A., Shaw A.T, & Iafrate A.J. (2016). Acquired Resistance to Crizotinib in NSCLC with MET Exon 14 Skipping. Journal of thoracic oncology : official publication of the International Association for the Study of Lung Cancer, 11(8).

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