Tcs sp5 2 dichroic cs confocal microscope

On the day of the last PCN injection, a vascular perfusion assay was performed by injecting 100 μl of DyLight® 488-labeled Lycopersicon esculentum (tomato) lectin (1 mg/ml, Vector Lab) intravenously 30 min prior to sacrifice. To check for vascular leakage, 100 μl of FITC-dextran (25 mg/ml, 70 kDa, Sigma-Aldrich) was intravenously injected into the mice 30 min before sacrifice. After the mice were perfused with PBS and 4% paraformaldehyde (PFA), the FITC-dextran or tomato-lectin in the tumor tissues were observed under a Leica TCS SP5 II Dichroic/CS confocal microscope (Leica, Wetzlar, Germany). The fluorescence intensity was measured using ImageJ software.

Retinas were incubated with or without anti-CCN1 antibody (Abcam) overnight. After washing several times, the samples were incubated for four hat RT with Alexa Fluor488-conjugated isolectin B4 (IB4, Invitrogen) and anti-rabbit Alexa Fluor 647-conjugated IgG. The whole-mount retinas were visualised and digital images obtained using a Leica TCS SP5 II Dichroic/CS confocal microscope (Leica, Wetzlar, Germany). The number of front tip cells with filopodia was counted in three different peripheral fields of the retina. The radial length of the blood vessels in the postnatal retina was measured as the shortest distance from the optic nerve head to the peripheral vascular front in each retinal quadrant. The vascular density in the whole-mounted retina was calculated as the IB4⁺ microvessel area divided by the total measured area of the retina and presented as a percentage.