Ab108307

Tissues were fixed in 10% normal buffer saline prior paraffin embedding. Four-micron tissue slices were deparaffinised and rehydrated. Epitope retrieval was performed at 60 °C for 20 min using Epitope Retrieval 2 (AR9640, Leica). Tissues were then stained with rabbit anti-CD46 at 0.078 μg/mL for 60 min (1:1000, ab108307, Abcam). Detection of primary antibody was performed using the Polymer Refine Detection Horseradish peroxidase for 8 min (DS9800, Leica). Between antibody or polymer incubation steps, tissues were washed twice with Wash buffer (AR9590, Leica). Tissues were counterstained with haematoxylin. Slides were processed using the Bond-Max (Leica).

Paraffin-embedded tissue blocks were cut into 2–3 μm sections and mounted on adhesive microscope glass slides. After deparaffinization and rehydration the antigen retrieval was performed in pre-warmed citrate buffer (pH 6 temp. 95°C) for 30 minutes. Sections were cooled to room temperature and incubated with a protein blocking serum-free solution for 15 minutes at 22°C to block non-specific binding.
For immunofluorescence staining, sections were separately incubated with three different antibodies: a monoclonal mouse anti-human CD31 antibody (Dako, JC70A, 1:50 dilution; a marker for vascular endothelium), a monoclonal rabbit anti-human CD46 antibody (Abcam, ab108307, 1:500 dilution), and a monoclonal murine anti-human SC5b-9 antibody (QUIDEL, A239, 1:250 dilution). Slides were incubated 30 minutes at pH 6 with the corresponding Alexa 546-conjugated antibody (anti-mouse or anti-rabbit; INVITROGEN Molecular Probe, diluted 1:1000). Reduction of the autofluorescence background was obtained by the incubation with Sudan Black B 0.1% (Sigma-Aldrich). Nuclei were stained with Prolong Gold antifade reagent with DAPI (INVITROGEN Molecular Probe). Slides were analysed by a Olympus BX61 microscope.