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Immunohistochemical Profiling of Germ Cells: MATERIALS AND METHODS(4)

METHODS(4)Double-Staining Using Fast Blue

Following incubation with the second primary antibody, sections were washed in TBS and then incubated for 30 min with the appropriate bio-tinylated secondary antibody diluted 1:500 in BSA/TBS. Sections were again washed in TBS, then incubated with avidin-biotin-alkaline phosphatase complex (DAKO); bound antibody was visualized with Fast Blue (Sigma). Single (DAB)-stained sections were counterstained with hematoxylin, dehydrated, and mounted with Pertex (Cell Path, Hemel Hempstead, U.K.); double-stained sections were mounted in Permafluor (Beckman Coulter, High Wycombe, U.K.). Both were visualized by light microscopy. buy claritin online


Immunofluorescence was used both for double-staining (CHK2/ MAGE-A4 and OCT4/MAGE-A4) and for triple-staining with antibodies against CHK2, MAGE-A4, and C-KIT. For the double- and triple-staining experiments, the antibodies were used at the following dilutions: anti-MAGE-A4, 1:30; anti-C-KIT, 1:100; anti-CHK2, 1:40; and anti-OCT4, 1: 1000. For double-staining with CHK2/MAGE-A4, the MAGE-A4 was visualized by Streptavidin-488 (Molecular Probes, Leiden, The Netherlands) via a biotinylated rabbit anti-mouse secondary antibody, whereas CHK2 was visualized by tyramide-enhanced Cy5 via an HRP-conjugated rabbit anti-mouse immunoglobulin G2a secondary antibody.