Isimiso Isingeniso:
Umgomo we-Multiplex Immunohistochemistry (mIHC) ukuthi i-antigen esezigabeni zethishu ibophezeleka ku-antibody eyinhloko. Bese, i-antibody yesibili ehlanganiswe ne-horseradish peroxidase (HRP) yengezwa ukuze ihlanganiswe ne-antibody eyinhloko. Ngemuva kokungeza i-tyramine fluorescence substrate, nama-substrates e-tyramine fluorescein avuliwe acushwa yi-HRP-catalyzed hydrogen peroxide. Ngemva kokwengezwa kwe-fluorescein substrate, i-tyramine fluorescein substrate engacushiwe yenziwa isebenze ngokuzikhandla yi-HRP phambi kwe-hydrogen peroxide. I-substrate esetshenzisiwe ibophezela ngokuhlanganyela ezinsalela zamaprotheni aseduze, okuhlanganisa i-tryptophan, i-histidine, ne-tyrosine, okuvumela ukubopha okuzinzile kwe-antigen-fluorescein.
Ekuhlolweni kokulebula nge-multiplex, amasosha omzimba ayisisekelo nesibili angaboshiwe asuka emzuliswaneni odlule angagezwa kusetshenziswa ukulungisa ukushisa noma i-antibody eluent. Ngokulandelayo, umzuliswano olandelayo we-primary-HRP amasosha omzimba wesibili uyafukanyelwa, ulandelwe ukusetshenziswa kwe-tyrosine fluorescein substrate ehlukile. Le nqubo ingaphinda ukuze kuzuzwe izinyathelo eziningi zokulebula. Njengoba i-antibody eyodwa kuphela efakwe ngesikhathi ohlelweni ngalunye, akukho ukukhathazeka mayelana nokuhlangana kwe-antibody cross-reactivity noma isidingo sokufanisa amasosha omzimba ayisisekelo nawesibili, kunciphisa kakhulu izinselele ezihambisana nokukhetha nokuqhathanisa amasosha omzimba avela ezinhlotsheni ezihlukene ekwakhiweni kokuhlola.
Ikhithi iqukethe odayi abathile be-tyramine fluorescent, okufaka okukodwa noma ngaphezulu kokulandelayo: TYR-480Plus, TYR-520Plus, TYR-570Plus, TYR-620Plus, TYR-690Plus, ne-TYR-780Plus. Laba bodayi be-fluorescent bangasetshenziswa ngabodwana noma behlanganiswe, okuvumela ukukhuliswa kwe-fluorescence okukodwa, okuphindwe kabili, okuphindwe kathathu, noma okuyinkimbinkimbi nakakhulu kanye nokulebula kwe-homologous antibody, okuthuthukisa kakhulu ukusebenzisana kwekhithi.
Eyethu I-Multi Fluorescence Staining Plus(mIHC) Kits okwamanje ingafinyelela imibala engu-9. Inhlanganisela yodayi ichazwe ngokuningiliziwe etafuleni elingezansi:
Izingxenye zekhithi:
I-TYR-***Plus Fluorescent Dyes, Isiphazamiso se-TSA, i-HRP yembuzi yokulwa negundane/unogwaja i-antibody yesibili, i-DAPI Staining Solution (Ilungele ukusetshenziswa), I-Antibody Diluent, 3% i-hydrogen peroxide.
Izinzuzo zeMulti Fluorescence Staining Plus(mIHC) Amakhithi:
1.Ukuzwela okuphezulu, izikhathi eziyi-10-50 eziphakeme kunezisetshenziswa ezijwayelekile zokuhlola ze-TSA,
2.Qaphela ukuthi ungabi nesignali enamandla kakhulu; Uma isignali iqine kakhulu, ukugxiliswa kukadayi/isikhathi sokusabela/ukugxiliswa kwe-antibody eyinhloko kuzoncishiswa,
3.Uma isignali iphansi kakhulu, khulisa ukugxiliswa kukadayi/isikhathi sokusabela/ukugxiliswa kwe-antibody eyinhloko;
4.Ukuphakama kokugxilisa kwe-Multi Fluorescence Staining Plus(mIHC) Kits, isignali ithuthukiswa kakhulu; I-1: 50 ingathola isignali enamandla kakhulu, futhi i-1:500 ilingana nesignali yekhithi evamile ye-TSA.
5.I-Multi Fluorescence Staining Plus(mIHC) Kit inokumelana okuqinile nokucisha odayi be-fluorescent, futhi ayidingi ukuchayeka elangeni kuyo yonke inqubo. Futhi ayidingi ukusebenza endaweni emnyama ngesikhathi sokusetshenziswa, kodwa ayikwazi ukuchayeka elangeni.
Inqubo yokwenza amabala:
Isibonelo sokusebenzisa izithombe wethuI-Multi Fluorescence Staining Plus Kits:
(A) Inkomba ye-fallopian tube tissue index A (570 yellow)+index B (520 green)+index C (690 powder);
(B) Inkomba yezicubu zezinso zegundane A (570 yellow)+index B (520 green)+index C (690 powder);
(C) Inkomba yesimila somdlavuza wesisu somuntu A (690 powder)+inkomba B (620 ebomvu)+inkomba C (570 ephuzi)+inkomba D (520 eluhlaza); (D) Inkomba yezicubu zamathumbu omuntu A (570 yellow)+index B (520 green)+index C (690 pink)+index D (620 red)+index E (480 green).
