imunologie - lucrari practice (3)
DESCRIPTION
Imunologie, UMFCV, Silosi Isabela; anul 3 medicina generalaTRANSCRIPT
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UNIVERSITATEA DE MEDICINĂ ŞI FARMACIE CRAIOVA
INTRODUCERE ÎN HISTOCHIMIE ŞI IMUNOHISTOCHIMIE
Partea a III-a
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PLANUL PREZENTĂRII
Metode de blocare a surselor de semnal fals in IHC
Detectia fluorescenta a semnalului
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PLANUL PREZENTĂRII
Metode de blocare a surselor de semnal fals in IHC
Detectia fluorescenta a semnalului
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Secvenţa tipică a IHC•Secţionare a blocului de parafină
•Clarificare (solvenţi organici)
•Hidratare (alcool în concentraţie descrescătoare)
•Spălare în apă
•Blocarea enzimelor endogene
•Spălare în sol. salină
•Blocarea situsurilor antigenice nespecifice
•Blocarea biotinei endogene
•Incubare cu primul anticorp
•Spălare în sol. salină
•Incubare cu anticorpul secundar şi sistemul de detecţie
•Detecţia semnalului
•Deshidratarea, clarificarea şi acoperirea cu lamela
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Blocarea enzimelor endogene-Peroxidaza-
• Peroxidazele contin gruparea HEM ce utilizeaza H2O2 ca acceptor de electroni pentru a cataliza oxidarea substratului (DAB, AEC).
• Peroxidaza exogene – extrasa din hrean.
• Peroxidazele endogene:– Hemoglobina (hematii)– Mioglobina (tesut muscular)– Citocromi (granulocite, monocite)– Catalze (ficat, rinichi)
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Blocarea enzimelor endogene-Peroxidaza-
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Blocarea enzimelor endogene-Peroxidaza-
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Blocarea enzimelor endogene-Peroxidaza-
Hemoragie cu reactie nespecifica.
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Blocarea enzimelor endogene-Peroxidaza-
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Blocarea enzimelor endogene-Peroxidaza-
Blocarea peroxidazei endogene se face prin suprasaturare cu H2O2 1-3% in apa distila sau metanol.
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Blocarea enzimelor endogene-Peroxidaza-
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Blocarea enzimelor endogene-Peroxidaza-
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Blocarea enzimelor endogene-Peroxidaza-
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Blocarea enzimelor endogene-Fosfataza alcalina-
• PA (intestin de vitel) muta o grupare P de pe un donor pe un acceptor (ambele componente ale substratului).
• PA endogena se gaseste in maduva hematogena, vezica urinara si intestin.
• Blocarea PA se face cu o solutie 5 mM de levamisol.
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Blocarea enzimelor endogene-Fosfataza alcalina-
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Blocarea biotinei endogene
• Biotina abunda in ficat, rinichi si tesutul limfoid.
• Vizualizarea biotinei endogene se acentueaza pe sectiunile tratate cu EDTA pH=9.
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Blocarea biotinei endogene
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Blocarea biotinei endogene
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Blocarea biotinei endogene
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Blocarea biotinei endogene
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Blocarea biotinei endogene
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Blocarea biotinei endogene
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Blocarea biotinei endogene
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Blocarea biotinei endogene
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Blocarea biotinei endogene
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Blocarea biotinei endogene
Biotina poate lega o singura data Avidina sau Streptavidina !!!
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Blocarea biotinei endogene
• Incubare cu streptavidina
• Spalare PBS
• Incubare cu biotina
• Spalare PBS
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Blocarea situsurilor antigenice nespecifice
• Se realizează pentru împiedicarea legării nespecifice a anticorpului primar care ar conduce la obţinerea unui semnal fals.
• Inainte de anticorpul primar, se incubează lamele 30 min la temperatura camerei cu ser normal al aceleiaşi specii cu anticorpul secundar, sau cu lapte degresat 1-3%.
• Nu se spala in PBS inainte de adaugarea anticorpului primar.
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Blocarea situsurilor antigenice nespecifice
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Blocarea situsurilor antigenice nespecifice
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Blocarea situsurilor antigenice nespecifice
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Blocarea situsurilor antigenice nespecifice
![Page 33: Imunologie - Lucrari Practice (3)](https://reader036.vdocuments.pub/reader036/viewer/2022081415/563db852550346aa9a929d9a/html5/thumbnails/33.jpg)
Secvenţa tipică a IHC•Secţionare a blocului de parafină
•Clarificare (solvenţi organici)
•Hidratare (alcool în concentraţie descrescătoare)
•Spălare în apă
•Blocarea enzimelor endogene
•Spălare în sol. salină
•Blocarea situsurilor antigenice nespecifice
•Blocarea biotinei endogene
•Incubare cu primul anticorp
•Spălare în sol. salină
•Incubare cu anticorpul secundar şi sistemul de detecţie
•Detecţia semnalului
•Deshidratarea, clarificarea şi acoperirea cu lamela
![Page 34: Imunologie - Lucrari Practice (3)](https://reader036.vdocuments.pub/reader036/viewer/2022081415/563db852550346aa9a929d9a/html5/thumbnails/34.jpg)
PLANUL PREZENTĂRII
Metode de blocare a surselor de semnal fals in IHC
Detectia fluorescenta a semnalului
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Detectia fluorescenta a semnalului
Anticorpii se conjuga cu fluorofori (cromofori) care excitaticu o lumina UV de o lungime de unda, pot emite o lumina UVcu energie mai joasa (cu o lungime de unda mai mare).
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Detectia fluorescenta a semnalului(in lumina reflectata!!!)
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Detectia fluorescenta a semnalului
Fluoroforii sunt de obicei compusi fenolici:Cei mai folositi: FITC (verde), Texas Red, CY3 (rosu),DAPI (albastru)
In ultimii ani au aparut nanocrostale care emit lumina pefrecvente ce depind de dimensiunile lor (nanocristale- Qdots)
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Detectia fluorescenta a semnalului
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Detectia fluorescenta a semnalului
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Detectia fluorescenta a semnalului
Avantajele IHC in fluorescenta:
1. Studiile de colocalizare sunt posibile numai prin fluorescenta.2. Microscopia confocala si deconvolutia permit colocalizarea si
reconstructia 3D reale.3. Detectia nu interfera cu structurile normale (semnal pur).4. Markerii fluorescenti se pot urmari si in vivo.
Dezavantaje:
1. Microscop special dotat cu filtre pentru fiecare cromogen.2. Vizualizarea nespecifica a unor compusi fluorescenti
(lipofuscina).
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Detectia fluorescenta a semnalului
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Detectia fluorescenta a semnalului
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Detectia fluorescenta a semnalului
CD31 – Alexa 488
Cd20cy – Alexa 594
Planuri optice reale
CD31 – Alexa 488
Cd20cy – Alexa 594
Deconvoluţie
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