Alexa488 anti mouse igg2a

To investigate the co-localization between macrophage-specific antigens, double immunofluorescence was carried out using antibody against MHC class II in combination with CD68 or CD163
antibody in fresh-frozen renal tissue sections (10 µm thick) from the control and CDDP-injected rats. Briefly, after fixation in acetone: methanol (1:1) for 10 min at 4°C, the sections were
incubated with 10% normal goat serum for 30 min. The sections reacted with the primary antibody overnight at 4°C. After rinsing with PBS, the sections were incubated for 45 min with the
following secondary antibodies: Alexa568/anti-mouse IgG1, Alexa488/anti-mouse IgG2a (Invitrogen Co., ×1,000). The sections were visualized with Vectashield mounting medium containing 4ˈ,
6-diamidino-2-phenylindole (DAPI; Vector Laboratories Inc.) for nuclear staining and analyzed using a virtual slide scanner (VS-120, Olympus, Tokyo, Japan).

Renal tissues from neonate and adult kidneys were collected and immediately immersed into mounting medium (TISSU MOUNT^®, Chiba Medical, Soka, Japan) and frozen on deep freezer. Sections were cut at 10 µm in thickness on a cryostat, thaw mounted onto MAS-coated micro slide glass (Matsunami Glass Ind., Ltd., Osaka, Japan). Double immunofluorescence was carried out using E-cadherin in combination with β-catenin, N-cadherin, aquaporin 1 and vimentin. Briefly, after fixation in cold acetone: methanol (1:1) for 10 min at 4°C, the sections were incubated with 10% normal goat serum for 30 min. The sections reacted with the primary antibody overnight at 4°C. After rinsing with PBS, the sections were incubated for 45 min with the secondary antibodies: Alexa568/anti-mouse IgG1, Alexa488/anti-mouse IgG2a (Invitrogen Co.; ×1,000). The sections were visualized with Vectashield mounting medium containing 40, 6-diamidino-2-phenylindole (Vector Laboratories) for nuclear staining, and captured by a laser scanning confocal imaging microscope (Nikon C1Si) and processed with EZ-C1 3.20 Free Viewer (Nikon, Tokyo, Japan).