Tcs sp2 acousto optical beam splitter confocal system

Fluorescence images were taken with Leica TCS SP2 Acousto-Optical Beam Splitter confocal system equipped with DMIRE2 inverted microscope (Diode 405 nm, Argon 488 nm, HeNe 594 nm; Leica Microsystems), Leica Confocal Software Version 2.61, Build 1537. Images were taken at room temperature (about 20 C) and files always exported as 8 bit format.

Formalin-fixed, paraffin-embedded tissue sections (5μm) were deparaffinized and either directly stained with hematoxylin and eosin (H&E) or immersed in an antigen retrieval solution (Citrate-EDTA buffer: 10mM Citric Acid, 2mM EDTA, 0.05% Tween-20, pH 6.2;) for 20 minutes. Frozen sections (10μm) fixed in acetone at −20°C for 10 minutes and evaporated at room temperature for 20 minutes. Sections were blocked for 30 minutes in 5% serum and incubated overnight with anti-human VE-Cadherin (1:40, C19, Sigma), anti-human PDGFRβ (1:40, AF385, R&D Systems), or anti–human CD31 (1:40, clone JC70A, Dako). Purified class- and species-matched IgGs (Vector Lab) were used as controls. Sections were incubated with fluorescein or Texas Red conjugated secondary antibody (Vector Laboratories). Slides were stained with the nuclear marker DAPI (Molecular Probe, Eugene, OR). Images were acquired using a Leica TCS sp2 Acousto-Optical Beam Splitter confocal system equipped with DMIRE2 inverted microscope camera (Leica Microsystems, Wetzlar, Germany).

Formalin-fixed, paraffin-embedded tissue sections (5 µm) of Hem 137 and Hem I-84 were deparaffinized and either directly stained with hematoxylin and eosin (H&E) or immersed in a retrieval solution (Citrate-EDTA buffer: 10mM Citric Acid, 2mM EDTA, 0.05% Tween-20, pH 6.2;) for 20 minutes at 95°C to 99°C. Sections were blocked for 30 minutes in 5% serum and stained with anti-human VE-cadherin and anti-human Plexin D1. For immunofluorescence staining, sections were incubated with appropriate FITC or Texas Red conjugated secondary antibody (Vector Lab). All slides were mounted using DAPI (Molecular Probe, Eugene) as a nuclear marker. Images were acquired using a Leica TCS sp2 Acousto-Optical Beam Splitter confocal system equipped with a DMIRE2 inverted microscope camera (Leica Microsystems).