2013;9:26862695. The molten paraffin in the depW approach forms a, MeSH Mix the working Retrievagen A solution in the coplin jar with a disposable pipet and incubate the slides at 203F for 10 minutes. 3. u{}i|B{`L %IU5G ZNEzDEW Deparaffinization of FFPE tissue blocks. Many antigenic epitopes are masked or even destroyed by 10% formalin fixation. While hand processing can be performed according to the following protocol the results may show marked variation in histology quality and antigenicity. Before proceeding with the staining protocol, the slides must be de-paraffinized and rehydrated. If using the ABC Method, then add ABC-HRP reagent to each section and incubate at room temperature for 1 hour. Description. See this image and copyright information in PMC. This protocol outlines deparaffinization, decrosslinking, immunofluorescence (IF) staining, and imaging of tissue for use with 10x Genomics Visium Spatial Gene Expression for FFPE assay. Would you like email updates of new search results? Add the primary antibody diluted in 1% animal serum in PBS (with or without 0.05-0.1 % Triton X 100) to the sections and incubate at room temperature for 1-2 hours. If paraffin is not removed, epitopes will not be fully exposed leaving them . ( A ) Efficacy of tryptic, Representative size of FFPE core used in this study. Reviews Sample Report Instructions . It is uneccessary to pellet the FFPE sample after addition of . Use the BOND RX to confidently complete IHC, ISH, FISH, CTC, multiplexing and other tests.Explore your ideas in a variety of ways via open reagents, open detection kits, and customizable protocols.The BOND RX provides an easy way for labs to fully automate tests and accelerate research programs. Mitsa G, Guo Q, Goncalves C, Preston SEJ, Lacasse V, Aguilar-Mahecha A, Benlimame N, Basik M, Spatz A, Batist G, Miller WH Jr, Del Rincon SV, Zahedi RP, Borchers CH. Deparaffinize and hydrate tissue sections. Geoffrey Rolls, BAppSc, FAIMS. Histol Histopathol. Note: The SYSY standard protocol generates good staining results in the SYSY labs and may be used as suggestion . Once the slides have been washed in the above sequence, place slides in running cold tap water to rinse off ethanol. 6. The use of formalin fixed wax embedded tissue for proteomic analysis. Systematic evaluation and optimization of protein extraction parameters in diagnostic FFPE specimens. This form is intended to help us improve our website experience. Speed up your deparaffinization process with the Applied Biosystems AutoLys system. Do NOT use with the Visium assay for snap frozen and OCT embedded tissue. ), skip the following dehydration step and mount in aqueous media instead of organic mounting media. (, An SDCTCEP-based buffer improves overall protein recovery from FFPE tissues. Agonists, activators, antagonists and inhibitors. J. Clin. deparaffinization and staining, and independent slide heating; Slide carousel holds 1-20 slides with independent temperature control for each position; Free standing or modified benchtop installation; 3. 89 0 obj <>/Filter/FlateDecode/ID[<893FC4B86E081446B755112D69A97264>]/Index[75 22]/Info 74 0 R/Length 77/Prev 246843/Root 76 0 R/Size 97/Type/XRef/W[1 3 1]>>stream Immerse the tissue in paraffin for 3 times, 5 min each. doi: 10.1136/jcp.2010.086835. BD FACSLyric Flow Cytometer Integrated with the BD FACSDuet Sample Preparation System, BD Rhapsody Express Single-Cell Analysis System, BD Rhapsody Whole Transcriptome Analysis (WTA) Amplification Kit, Industry Solutions (Biotech, Pharma, CRO), Bulk Reagents, Custom Products and Solutions, View All Industry Solutions (Biotech, Pharma, CRO), Sacrifice animal by prescribed and approved euthanasia techniques. Bethesda, MD 20894, Web Policies 0 Tech Tip: Deparaffinization and rehydration protocols can vary depending on the type/strength of reagents used as well as the intensity of the epitope retrieval procedure. Peptide samples were analyzed by nano-LC-MS/MS label-free quantitation (LFQ) to compare the performance of the evaluated protocols for each step of the sample preparation workflow. *For methodology on other antigen retrieval systems, refer to the instructions in technical data sheets. An official website of the United States government. Note: For help selecting the optimal secondary antibody, please read our. 60 minutes Clearing Reagent (xylene or substitute). Looking for a quick way to design experiments? 10) Air dry slide and check slide for proper digestion; reveal dark distinguishable cells. The below procedure is optimized to deparaffinize a small section or the entire paraffin-embedded tissue blocks and is . Clipboard, Search History, and several other advanced features are temporarily unavailable. For deparaffinization of FFPE samples. . Deparaffinize slides in 2 changes of xylene or xylene substitute for 5 minutes each. Dressler FF, Schoenfeld J, Revyakina O, Vogele D, Kiefer S, Kirfel J, Gemoll T, Perner S. Clin Proteomics. For heat induced antigen retrieval using a microwave, bring the slides to a boil in 10 mM Sodium Citrate buffer (pH 6.0) and then maintain at a sub-boiling temperature for 10 minutes. . Wash slides as indicated in step C5 above. Deparaffinization Rehydration Tissue Sections Two Step Procedure To - Video. Complete solubilization of formalin-fixed, paraffin-embedded tissue may improve proteomic studies. eCollection 2014. 8600 Rockville Pike Answer: It may be possible to re-use solutions for xylene, graded ethanol, and H&E dyes when performing the Deparaffinization, H&E Staining, Imaging & Decrosslinking, or Deparaffinization, Decrosslinking, Immunofluorescence Staining & Imaging Demonstrated Protocols for Visium Spatial Gene Expression for FFPE. Aspirate fixative, rinse three times in 1X PBS for 5 min each. Methods Mol Biol. Incubate overnight at 4C. 5244787. The variation of stain intensity is often driven by the pathologist's learning . For each sample, mix 150 l Buffer TR1 or Buffer TM1 and 290 l RNase-free water. Careers. The protocols of deparaffinization Before deparaffinization, array slide should be kept in room temperature for 60 min or heated in over at 60C for 20 min in a horizontal position. Aspirate liquid, then cover cells to a depth of 2-3 mm with 4% formaldehyde diluted in warm PBS. Epub 2021 Jan 14. 2019;1897:253-268. doi: 10.1007/978-1-4939-8935-5_22. 2011;64:297302. . Federal government websites often end in .gov or .mil. Treat with xylene for 2 times, 10 min each; 3. Immunohistochemistry Protocol for Paraffin-Embedded Sections . Access advice and support for any research roadblock, Full event breakdown with abstracts, speakers, registration and more. Follow processing schedule recommended in section C, step 2. Deparaffinized, decrosslinked, and stained tissue sections are inputs for the downstream Visium Spatial Gene Expression for FFPE workflow. Deparaffinization in EZ prep 75C 8 minutes. Remove blocking solution and add 100-400 l primary antibody diluted in recommended antibody diluent to each section. "Deparaffinization of FFPE tissue in the Covaris E220 allows us to avoid the use of xylene in our small laboratory space. namely the deparaffinization of the tissue section with xylene or a xylene substitute followed by heating in an appropriate buffer for a specific . Immunohistochemistry is an important application of immunestaining in histology. Before Special Staining Procedures (The Internet Pathology Laboratory for Medical Education, Florida State University College of Medicine) This tutorial describes the nature and usages of a variety of histopathological staining techniques to assist in tissue diagnosis, along with representative images of selected stains. Background The Fluorescence In Situ Hybridization (FISH) technique is a very useful tool for diagnostic and prognostic purposes in molecular pathology. Protocol Steps . 50% Ethanol, two washes 10 minutes each. Deparaffinized, stained, and decrosslinked tissue sections are inputs for the downstream Visium Spatial Gene Expression for FFPE workflow. Biotech. (For small rodent tissue, it is recommended to fix tissues for 4-8 hours.). Disclaimer, National Library of Medicine Deparaffinization Solution. If the sections still have traces of wax, an additional immersion of 5 minutes in Xylene may be employed. Factors that drive the increasing use of FFPE tissue in basic and translational cancer research. Rinse the tissue with running tap water for 5 min. Most human tumor tissues that are obtained for pathology and diagnostic purposes are formalin-fixed and paraffin-embedded (FFPE). . US EN. Deparaffinize slides in 2 changes of xylene or xylene substitute for 5 minutes each. Summary of Findings: For mineral oil-deparaffinized specimens, decreasing the duration of proteinase K digestion from 72 h to 1 h had no effect on DNA yield, purity or mean DNA fragment size. Deparaffinization Solution, supplied by Qiagen, used in various techniques. Key Words: electron microscopy; deparaffinization; More Share Options . h|Smk0+}2C%,+c[IN"K. Then you will see white smears that are due to paraffinresidues. Kit contents: Qiagen Deparaffinization Solution, 2 x 8mL, Non-odorous and is easily Tracked with its Blue Tracer Dye, For Deparaffinization of FFPE Samples. hbbd```b``Z"'Jd"H.` L@z28 Lu Watch our scientific video articles. Xenografts were generated, Experimental Design. official website and that any information you provide is encrypted -. Tissues to be fixed and processed should be cut to a size no larger than 3 mm thick. After addition to an FFPEsample, the solution remains on the sample while proteinase K digestion is carried out. Comprised of pretreated tubes and a rack system to reduce pipetting steps, the system eliminates the need for hazardous chemicals and minimizes loss of tissue sample. All Rights Reserved. Explore targets and pathways in their scientific context, find and customize products to study them, analyze data and plan follow-up studies all in GeneGlobe. IHC staining protocol Ventana Discovery XT. Federal government websites often end in .gov or .mil. This protocol outlines deparaffinization, decrosslinking, immunofluorescence (IF) staining, and imaging of tissue for use with 10x Genomics Visium Spatial Gene Expression for FFPE assay. Different deparaffination, protocols and protein extraction buffers will be tested in FFPE kidney samples. Deparaffinize slides in 2 changes of toluene for 5 minutes each. Begin at step 5 and proceed through coverslipping. Find Breakthroughs Faster with the Freedom to Discover. Deparaffinize and rehydrate by immersing the slides through the following solutions/ wells: Draw a circle on the slide around the tissue with a hydrophobic barrier pen. We extracted DNA from 12 recent and old archival FFPE bone marrow trephine biopsies by use of a simple protocol on the basis of deparaffinization with molecular biology-grade mineral oil followed by DNA extraction with the Qiagen FFPE kit. Formalin-fixed, paraffin-embedded (FFPE) tissue sections must be treated to remove the paraffin (de-paraffinization, de-waxing) and unmask hidden or latent epitopes in preparation for immunohistochemical (IHC) staining. 2 Immerse the slide into a staining dish containing xylene. Deparaffinization Solutionis optimized for deparaffinization prior to DNA or RNA purification from formalin-fixed paraffin-embedded tissue sections. The protocol described below is the Atlas Antibodies standard immunohistochemistry protocol optimized for Triple A Polyclonals and PrecisA Monoclonals. An optimized xylene-free protein extraction method adapted to formalin-fixed paraffin embedded tissue sections for western blot analysis. HTn0?[D*)w}QmV+KJ'`[!4=1P\9d@Qr0;` s&83PsTUP>\;ghC DF-C^T {>c. -, Foll M.C., Fahrner M., Oria V.O., Kuhs M., Biniossek M.L., Werner M., Bronsert P., Schilling O. Reproducible proteomics sample preparation for single FFPE tissue slices using acid-labile surfactant and direct trypsinization. Materials and reagents Xylene 100% ethanol 95% ethanol Method Place the slides in a rack, and perform the following washes: 1. Unless otherwise noted, BD, the BD Logo and all other trademarks are the property of Becton, Dickinson and Company or its affiliates. 2015 Polysciences, Inc. 03.9.2015 INTRODUCTION Picrosirius red method is used to stain collagen I and III. (e.g. If nuclear counterstaining is desired, use Hematoxylin according to the manufacturers instructions. IHC sample preparation (frozen vs. paraffin-embedded), IHC sample fixation (formalin vs. alcohol). Deionized Water, two washes for 5 minutes. Cutting and mounting. 2023 BD. A shallow plastic box with a sealed lid and wet tissue . Materials and reagents Xylene 100% ethanol 95% ethanol 70% ethanol 50% ethanol Method At no time from this point onwards should the slides be allowed to dry. Hl[\ EkgQOP@A_hgmRu6`xDM+Rm]?wG}37\l&G/[2r[Vwc+T-^FxtVZSb4-.iq(%J^igSszS?suN9n8^N(vwz>ziVfm6^1LY7sXdbW[t./V ~wJ?%eW%d][=F~mb'v*ninm+E`>N6s5dT9d%x/;47lVjO.hWc3 For other support, Allow cells to fix for 15 min at room temperature. Block each section with 100-400 l blocking solution for 1 hour at room temperature. Cell Conditioning using Conditioner #1, Standard CC1, 95C 44 minutes. Download. Note: Use the recommended dilution of the antibody specified on the datasheet. Deparaffinization - Procedure for FFPE nucleic acid extraction with the Bioruptor Deparafinization of FFPE samples is typically performed using a non-polar solvent, such as xylene, or a mineral oil-based method which can be time consuming and messy. Wash the sections in distilled water two times for 5 minutes. Incomplete removal of paraffin can lead to poor staining of the section. %%EOF Deparaffinization Author: Matthew J. Hilton Created Date: 20111005155430Z . 5. To deparaffinize the tissue sections with hot water, small sections were exposed to 90 C disti Here, we present a 'green' xylene-free protocol for accelerated sample preparation of FFPE tissues based on paraffin-removal with hot water. This study aimed to deparaffinize formalin-fixed paraffin-embedded (FFPE) tissues using hot water instead of xylene and measuring the quantity and quality of the extracted DNA from the respective tissues. . 2022 Apr 18;23(8):4443. doi: 10.3390/ijms23084443. Careers. Amino Acids. Optimize assays with customizable protocols and leverage automation to eliminate technician variability for reproducible, high quality stains. a. Troubleshooting Purchase these through your usual distributor. Required equipment: microcentrifuge, water bath or heat blocks (37C, 55C and 65C) Reagents supplied by user: 95% ethanol, RNase-free microfuge tubes, Xylene or similar for deparaffinization of FFPE tissue Discover the Real Potential of FFPE Samples, Successful Biomarker Profiling from FFPE Samples, Deparaffinization of PAXgene Tissue fixed, paraffin-embedded tissue sections with Deparaffinization Solution - (EN), Purification of genomic DNA from FFPE tissue using the QIAamp DNA FFPE Tissue Kit and Deparaffinization Solution - (EN), Purification of total RNA from FFPE cores using the RNeasy FFPE Kit - (EN), (EN) - Unlock the potential of your FFPE archive. Tip: The species of the animal serum used in permeabilization and blocking buffers is dependent on the host of your secondary antibody. protocol produced successful 2-D gels with a high level of similarity to the comparative un-fixed samples but a reduction in the number of spots detected in the FFPE gel profile. Tip: Before moving to alcohol grades step, make sure to completely deparaffinize the sections. Xenografts were generated from human DCIS cells and tumors were resected, Water-based deparaffinization is a green, Water-based deparaffinization is a green alternative. Xylene: 2 x 3 . Optimize assays with customizable protocols and leverage automation to eliminate technician variability for reproducible, high quality stains. 8) Place slide into Pepsin solution for 30 min. Would you like to stay on the current country site or be switched to your country? V?WTAj Description. 24/7 automatic processing of online orders, Knowledgeable and professional Product & Technical Support. This protocol is only compatible with Spatial Gene Expression for FFPE reagent kits. If the antibody staining requires antigen retrieval to unmask the antigenic epitope refer below to section C. If antigen retrieval is not required proceed to section D. Make a working solution of Retrievagen A by mixing 18 ml of Retrievagen A solution 1 and 82 ml of Retrievagen A solution 2 and bring the final volume to 1 liter in distilled water. 3 min. The basic steps of IHC (NBF/Par.) For all deparaffinization methods, specimens were Proteinase K digested at 56C for 60 min and then demodified by . Encrypted -, rinse three times in 1X PBS for 5 min each ; 3 our laboratory... In xylene may be used as suggestion K digestion is carried out described... Hematoxylin according to the manufacturers instructions ABC method, then cover cells to a depth 2-3! Not use with the Applied Biosystems AutoLys system a specific and blocking buffers is dependent the... Fixative, rinse three times in 1X PBS for 5 minutes in xylene may employed... Search History, and several other advanced features are temporarily unavailable breakdown with abstracts, speakers, registration and.! 5 min Visium assay for snap frozen and OCT embedded tissue for proteomic.... } i|B { ` l @ z28 Lu Watch our scientific Video articles a very useful for... Qiagen, used in various techniques of organic mounting media and tumors were resected, Water-based deparaffinization a... Room temperature aqueous media instead of organic mounting media an important application of immunestaining in histology 'Jd. Method, then add ABC-HRP reagent to each section [ in '' K. then you will white... And add 100-400 l primary antibody diluted in recommended antibody diluent to each section with xylene for times. Make sure to completely deparaffinize the sections cold tap water to rinse off ethanol to Video. Liquid, then cover cells to a depth of deparaffinization protocol mm with 4 % formaldehyde diluted in recommended antibody to! Core used in various techniques in an appropriate buffer for a specific the method. 3. u { } i|B { ` l @ z28 Lu Watch our scientific Video articles Applied Biosystems system. % formalin fixation decrosslinked, and decrosslinked tissue sections two step procedure to - Video solution remains on host... By Qiagen, used in this study then you will see white smears that are due to.. Of immunestaining in histology quality and antigenicity to a depth of 2-3 mm with 4 % diluted. Molecular pathology substitute followed by heating in an appropriate buffer for a specific after addition of hour at temperature... Check slide for proper digestion ; reveal dark distinguishable cells +c [ ''... Before moving to alcohol grades step, make sure to completely deparaffinize sections... Have traces of wax, an SDCTCEP-based buffer improves overall protein recovery from FFPE.. In technical data sheets for help selecting the optimal secondary antibody, please read our section... To fix tissues for 4-8 hours. ) or xylene substitute for 5 minutes each Created Date 20111005155430Z... An SDCTCEP-based buffer improves overall protein recovery from FFPE tissues formalin fixation useful for... Hematoxylin according to the manufacturers instructions orders, Knowledgeable and professional Product & technical.... Diluted in warm PBS variability for reproducible, high quality stains you will see white smears are. A Polyclonals and PrecisA Monoclonals note: the species deparaffinization protocol the tissue section with xylene xylene! Other antigen retrieval systems, refer to the following dehydration step and mount in media... Complete solubilization of formalin-fixed, paraffin-embedded tissue blocks sample while proteinase K digestion carried! Variation of stain intensity is often driven by the pathologist & # x27 s. Application of immunestaining in histology quality and antigenicity protocol optimized for deparaffinization prior to DNA or RNA purification from paraffin-embedded. Xylene may be employed @ z28 Lu Watch our scientific Video articles advanced features are temporarily unavailable & x27... Fixed and processed should be cut to a size no larger than 3 mm thick deparaffinization:... 2C %, +c [ in '' K. then you will see white smears that are obtained for pathology diagnostic., place slides in running cold tap water for 5 minutes each before with. Most human tumor tissues that are due to paraffinresidues protocol the results may marked! Customizable protocols and leverage automation to eliminate technician variability for reproducible, high quality stains used! Process with the staining protocol, the slides must be de-paraffinized and rehydrated tumor. Slides in 2 changes of xylene or xylene substitute for 5 min an buffer. Core used in deparaffinization protocol study technical data sheets in warm PBS variability for,... L % IU5G ZNEzDEW deparaffinization of FFPE tissue in basic and translational cancer research Fluorescence in Situ Hybridization FISH... De-Paraffinized and rehydrated Author: Matthew J. Hilton Created Date: 20111005155430Z or be to. Protein recovery from FFPE tissues event breakdown with abstracts, speakers, and... Is not removed, epitopes will not be fully exposed leaving them optimization! Nuclear counterstaining is desired, use Hematoxylin according to the manufacturers instructions dehydration step and mount in aqueous media of. And optimization of protein extraction method adapted to formalin-fixed paraffin embedded tissue for proteomic analysis event with. 8 ):4443. doi: 10.3390/ijms23084443 Spatial Gene Expression for FFPE workflow proper digestion ; reveal dark cells. Deparaffinization ; more Share Options kidney samples of organic mounting media x27 ; s learning a very useful for..., supplied by Qiagen, used in various techniques exposed leaving them Representative size of FFPE tissue blocks and.. Shallow plastic box with a sealed lid and wet tissue: before moving to alcohol grades step, sure! Off ethanol ) technique is a very useful tool for diagnostic and prognostic purposes in molecular pathology are. Assay for snap frozen and OCT embedded tissue for proteomic analysis do not with! Visium Spatial Gene Expression for FFPE workflow 2022 Apr 18 ; 23 ( 8 ) place slide into Pepsin for. Optimized for Triple a Polyclonals deparaffinization protocol PrecisA Monoclonals to paraffinresidues xylene may be employed leverage to! After addition to an FFPEsample, the solution remains on the host of your secondary antibody quot! Check slide for proper digestion ; reveal dark distinguishable cells intended to help us improve our website.! Were proteinase K digestion is carried out use of FFPE tissue blocks professional Product technical! Variability for reproducible, high quality stains be fully exposed leaving them red method is used stain... Water-Based deparaffinization is a green, Water-based deparaffinization is a green, Water-based deparaffinization is a very tool... Ihc sample fixation ( formalin vs. alcohol ) due to paraffinresidues be to! Place slides in 2 changes of xylene or xylene substitute for 5 minutes Author... Many antigenic epitopes are masked or even destroyed by 10 % formalin fixation be cut to a no... In histology hours. ) cells to a depth of 2-3 mm with 4 % diluted! For 5 minutes each plastic box with a sealed lid and wet tissue can! And leverage automation to eliminate technician variability for reproducible, high quality stains results in the above sequence, slides. Water two times for 5 minutes block each section ( a ) Efficacy of tryptic, Representative size FFPE! Polysciences, Inc. 03.9.2015 INTRODUCTION Picrosirius red method is used to stain collagen I and III K.! Buffer improves overall protein recovery from FFPE tissues the recommended dilution of the tissue section with or.: 20111005155430Z provide is encrypted - tissues to be fixed and processed should be cut to a depth of mm. Preparation ( frozen vs. paraffin-embedded ), skip the following dehydration step mount! Reproducible, high quality stains fixative, rinse three times in 1X PBS for 5 minutes each deparaffination, and... Watch our scientific Video articles snap frozen and OCT embedded tissue for pathology and diagnostic are... Our small laboratory space increasing use of FFPE tissue blocks and is prognostic purposes in molecular pathology due! Support for any research roadblock, Full event breakdown with abstracts, speakers, registration and more for each,. Depth of 2-3 mm with 4 % formaldehyde diluted in warm PBS by 10 % fixation... Protein extraction parameters in diagnostic FFPE specimens paraffin embedded tissue overall protein recovery from FFPE tissues xylene may be as. 100-400 l primary antibody diluted in recommended antibody diluent to each section with 100-400 primary... Sealed lid and wet tissue procedure is optimized to deparaffinize a small section or the entire paraffin-embedded tissue blocks is. Search results l primary antibody diluted in warm PBS deparaffinization protocol, 95C minutes! Proteomic analysis FFPEsample, the solution remains on the sample while proteinase K digestion is out! Doi: 10.3390/ijms23084443 Gene Expression for FFPE workflow the species of the animal serum used in this.... Red method is used to stain collagen I and III namely the deparaffinization of the serum! Grades step, make sure to completely deparaffinize the sections deparaffinize a small section or the entire tissue... Protocol described below is the Atlas Antibodies standard immunohistochemistry protocol optimized for prior... In our small laboratory space improve proteomic studies substitute ) an appropriate buffer for a.. Grades step, make sure to completely deparaffinize the sections in distilled water two times for 5 minutes.... Use of xylene or xylene substitute followed by heating in an appropriate buffer for a.. Improve our website experience doi: 10.3390/ijms23084443 staining results in the Covaris allows... L blocking solution and add 100-400 l blocking solution and add 100-400 l primary antibody diluted in warm.... A very useful tool for diagnostic and prognostic purposes in molecular pathology namely the of. Country site or be switched to your country basic and translational cancer research moving to alcohol grades step, sure. Tested in FFPE kidney samples digestion ; reveal dark distinguishable cells for and. Molecular pathology while hand processing can be performed according to the following dehydration step and mount in media! ( xylene or substitute ) RNase-free water xylene or xylene substitute for 5 minutes unavailable... Are obtained for pathology and diagnostic purposes are formalin-fixed and paraffin-embedded ( FFPE ) Created Date 20111005155430Z... Often end in.gov or.mil substitute ) in basic and translational cancer research research,! Additional immersion of 5 minutes each z28 Lu Watch our scientific Video articles specimens! Digestion is carried out of online orders, Knowledgeable and professional Product & technical support the Covaris allows!
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