6.2 Reagenzien für die Immunhistologie
6.2.1 Phosphatgepufferte Kochsalzlösung
7,8g NaH2PO4 (Fa. Fluka, Buchs, CH, Art.Nr. 71496) werden in 5 Liter destilliertem Wasser gelöst und mit
1M Natronlauge (Fa. Merck, Darmstadt, Art.Nr. 9137) auf pH 7,1 eingestellt.
6.2.2 Demaskieren der Antigene mit Demaskierungslösung G
Demaskierungslösung G (Fa. Biologo, Kronshagen, Art.Nr.: DE-007) in einer Verdünnung von 1:4 mit PBS-Puffer ansetzen.
Die Schnitte 30 Minuten bei einer Temperatur von 92-95Æ C im Wasserbad inkubieren.
6.2.3 Bleichen von Melanin mit Wasserstoffperoxid
Entparaffinieren der Schnitte bis Aqua dest., verbringen für 24 bzw. 48 h in 10%iger H2O2 -Lösung.
6.3 ABC-Methode der immunhistochemischen Färbungen
I. Entparaffinieren und Rehydrieren
Xylol 1: 5 Minuten, Xylol 2: 5 Minuten, Xylol 3: 5 Minuten, Isopropanol: 5 Minuten, Etha-nol 96%: 2-3 Minuten.
II. Hemmung der endogenen Peroxidase
197ml Ethanol 85% + 3ml 30% Perhydrol (Inkubationszeit 30 Minuten)
III. Spülen mit PBS-Puffer (Inkubationszeit 3x5 Minuten)
IV. Wechsel von Küvetten in Coverplates (Shandon GmbH, Frankfurth/Main) und erneutes Spülen mit PBS-Puffer (Inkubationszeit 2x5 Minuten)
V. Inkubation mit dem Blockserum in Gebrauchsverdünnung (inaktiviertes Normalserum der Spezies, aus der der Sekundärantikörper gewonnen wurde, zur Unterdrückung von unspezifischen Hintergrundfärbungen, Inkubationszeit 20 Minuten)
VI. Inkubation mit dem primären Antikörper in Gebrauchsverdünnung mit PBS-Puffer (In-kubationszeit: über Nacht bei -4ÆC)
VII. Spülen mit PBS-Puffer (Inkubationszeit 3x5 Minuten)
VIII. Überschichten mit dem sekundären, biotinylierten Antikörper in Gebrauchsverdün-nung (Inkubationszeit 30-45 Minuten)
IX. Spülen mit PBS-Puffer (Inkubationszeit 3x5 Minuten)
X. Inkubation mit dem Avidin-Biotin-Immunperoxidase-Komplex in Verdünnung von 1:250 mit PBS-Puffer (Inkubationszeit 30-60 Minuten)
XI. Wechsel von Coverplates in Küvetten, Spülen mit PBS-Puffer (Inkubationszeit 3x5 Minuten)
XIII. Spülen mit PBS-Puffer (Inkubationszeit 3x5 Minuten)
XIV. Verbringen in fließendes Leitungswasser (Inkubationszeit 15 Minuten) XV. Gegenfärben mit Hämalaun nach Meyer (Inkubationszeit 1,5 Minuten) XVI. Bläuen unter fließendem Leitungswasser (Inkubationszeit 10-15 Minuten) XVII. Eindecken der Schnitte mit Kaisers Glyceringelantine
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Kleintierprax. 36, 203-205