Digital analyse af immun farvning af ZW10 interagerende protein i humant lunge væv
Summary
ZW10 interagerende protein (ZWINT) deltager i mitotisk spindel check point og patogenesen af carcinoma. Her introducerer vi en metodologi for immun farvning af ZWINT i humant lungekræft væv, efterfulgt af den digitale scanning af hele dias og billedanalyse. Denne metode kan levere digitale billeder af høj kvalitet og pålidelige resultater.
Abstract
Formålet med denne undersøgelse er at indføre en metodologi for immun farvning af humant lunge væv, efterfulgt af hele dias digital scanning og billedanalyse. Digital scanning er en hurtig måde at scanne en stak af slides og producere digitale billeder med høj kvalitet. Det kan producere samstemmende resultater med konventionel lys mikroskopi (CLM) af patologer. Desuden gør tilgængeligheden af digitale billeder det muligt, at det samme dias kan observeres samtidigt af flere mennesker. Desuden kan digitale billeder af dias gemmes i en database, hvilket betyder, at den langsigtede forringelse af glas glider undgås. Begrænsningerne af denne teknik er som følger. For det første har det brug for forberedt væv af høj kvalitet og de originale Immunhistokemi (IHC) slides uden nogen skade eller overskydende tætningsmiddel rester. Anden, tumor eller nontumor områder bør specificeres af erfarne patologer før analysen ved hjælp af software, for at undgå enhver forvirring om tumoren eller nontumor områder under scoring. For det tredje skal operatøren styre farvegengivelsen i hele digitaliseringsprocessen i hele slide Imaging.
Introduction
ZW10 interagerende protein (zwint) er en nødvendig bestanddel af kinetochore-komplekset, som er involveret i den mitotiske spindel checkpunkt1,2,3. Det er blevet rapporteret, at nedbrydningen af zwint fører til afvigende for tidlig kromosom adskillelse1,2,3. Nylige undersøgelser har antydet, at zwint er involveret i patogenesen af flere tumorer ved at fremme udbredelsen af tumorceller4,5. Vi har tidligere rapporteret overekspression af ZWINT i lungekræft5. Det er blevet almindeligt accepteret, at analysen af dias af patologer ved hjælp af CLM er tidskrævende og ikke kvantitativ6,7,8. Desuden kan forværringen af lagrede glas slides gøre det umuligt at trække tidligere oprettede slides. Den nye metode til computerbaseret digital helslide Imaging (wsi) kan overvinde disse begrænsninger6,7,8.
Til dette formål beskriver vi en metodologi for immun farvning af ZWINT i humant lungekræft væv, kombineret med hel-slide digital scanning og software-baseret billedanalyse. Den største fordel ved denne metode er at fremstilling af konkordant resultater med CLM. Denne teknologi kan anvendes i vid udstrækning i områder med patologisk scoring af hematoxylin-eosin farvning (H & E) og IHC, fluorescens in situ hybridisering (fisk), vævs mikroarrays (TMA), og Drug opdagelse og udvikling.
Protocol
Representative Results
Discussion
Hel-slide scanning er ved at blive et varmt emne for sin robuste scanning og produktion af høj kvalitet billeder til kliniske og forskningsformål11,12,13. Billeder kan produceres ved slide-scanning mikroskoper inden for minutter11,12,13. Ved at anvende denne metode, vi opnåede billeder af høj kvalitet til ZWINT IHC slides og sammenl…
Declarações
The authors have nothing to disclose.
Acknowledgements
Dette projekt blev støttet af Kinas nationale naturlige fundament (nr. 81500151, 81400121, 81270607, 81541027 og 81501352) og det naturlige fundament for Hubei-provinsen (Kina) (no. 2017CFB631). Forfatterne udtrykker deres påskønnelse af Guo Qin, Chang min, Li Hui, og deres kolleger på Wuhan Google Biological Technology Co., LTD for deres tekniske support. Forfatterne takker også Muhammad Jamal for sprog redningen.
Materials
| Pannoramic MIDI | 3D HISTECH | Cat: PMIDI-040709 | An automated digital slide scanner with a remarkable feature set :12-slide capacity, fluorescence scanning, and many more. |
| QuantCenter | 3D HISTECH | Downloaded from the official website of the company | The framework for 3DHISTCH's image analysis applications. |
| LEICA RM2235 | Leica Microsystems | Cat: 14050038604 | The enhanced precision of the new accessories will add convenience to block to knife approach as well as specimen orientation. |
| Rabbit anti-human Anti-ZWINT antibody | Abcam | Cat: ab197794 | Immunohistochemical analysis of ZWINT in human lung tissue. |
| Anti-rabbit secondary antibody | Wuhan Goodbio Technology | Cat:GB23303-1 | Secondary antibody for IHC staining. |
| Phosphate-buffered saline | Wuhan Goodbio Technology | Cat:G0002 | A solution containing a phosphate buffer. |
| OLYMPUS CX23 | OLYMPUS | Cat:6M87620 | Microscope for detection of H&E or IHC slides. |
| Dimethylbenzene | Shanghai Lingfeng Chemical Reagent | Cat:1330-20-7 | A colorless, flammable fluid used as a solvent and clarifying agent in the preparation of tissue sections for microscopic study. |
| Hematoxylin Staining Solution | Wuhan Servicebio technology | Cat:G1039 | It is commonly used for histologic studies, oftern colors the nuclei of cells blue. |
| Tween 20 | Baitg | Cat:2005-64-5 | It is a polysorbate-type nonionic surfactant formed by the ethoxylation of sorbitan before the addition of lauric acid. It is used as a deterent and emulsifier in pharmacological applications. |
| Citric acid repair liquid | Wuhan Servicebio technology | Cat:G1202 | Is is used to repair antigen after fixation during IHC procedure. |
| LEICA ASP200s | Leica | Cat: 14048043626 | It was designed for routine and research histopathology of up to 200 cassettes. |
| LEICA Arcadia H | Leica | Cat: 14039354103 | It is a heated paraffin embedding station and allows for simple operation and precise control, resulting in improved quality, a smooth workflow and reliability. |
| LEICA Arcadia C | Leica | Cat: 14039354102 | It is a cold plate holding more than 60/65 cassettes on its large working surface. It was designed with an environment adaptive control module to make sure the operating temperature is always stabilized at -6°C. |
| CaseViewer Software | 3DHISTECH |
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