Axiocammr3 camera

Fixed brains were cut into 40 μm sections using a Vibratome (Microm, France). Sections were permeabilized with 0.1% Triton X-100 in PBS, 2% Bovine Serum Albumin (BSA) and then incubated for 48h at 4°C with primary antibody (anti-IBA1 1:2000, Wako; anti-GFAP 1:500, Dako). After three washes in PBS, sections were incubated with the appropriate secondary antibody. Wide field or Apotome images of the whole unilateral hippocampus were acquired using an Imager Z1 microscope (Zeiss) equipped with an AxioCam MR3 camera. For analysis of IBA1 staining, for each field, 11 images corresponding to 10 μm-thick optical sections were acquired. For analysis of GFAP staining, for each field, 5 images corresponding to 4 μm-thick optical sections were acquired. Stacked images were merged and analyzed through the use of a custom developed ImageJ macro to determine (1) the number of immunopositive cells and mean cell body size (IBA1 staining) and (2) the percentage of immunopositive cell surface (GFAP staining). Fluoro-jade staining was performed according to manufacturer recommendations. Slices were examined with Imager Z1 microscope (Zeiss). The presence of positively stained neurons was observed in 6 sections randomly selected from the rostral and caudal portions of the hippocampus.

For microscopy, cells were transferred to a microscopy slide coated with a thin film of 1% (w/vol) agarose (Roth). Fluorescence microscopy was carried out at room temperature using a Zeiss Axio Observer Z1 microscope (Zeiss, Jena, Germany) equipped with an HXP 120 V light source (Zeiss, Jena, Germany), an αPlan-APOCHROMAT 100×/1.46 oil immersion objective (Zeiss, Jena, Germany) and an Axio Cam MR3 camera (Zeiss, Jena, Germany). Visualization of mCherry was achieved using Carl Zeiss filter set 64 HE (574–599 nm excitation, 605 nm beam splitter and 612–682 nm emission). Image acquisition and analysis were performed with Zen 2 software (Zeiss, Jena, Germany).

The anti-angiogenic properties of the compounds were determined using the Matrigel™ assay. Twenty-four-well plates were coated at 4°C with 250 μl of Matrigel™ solution (1:1 dilution in cell culture medium) and were allowed to solidify at 37°C for 1 h before seeding. HMEC-1 cells were then seeded at 100,000 cells per well and allowed to adhere for 5 min before treatment was initiated. HMEC-1 cells were treated with 10 μM of compounds. Photographs were taken after 8 h using the 5 × objective of an Axiovert 200M fluorescent microscope coupled to an AxioCamMR3 camera driven by AxioVision 4.8 software (Carl Zeiss, North Ryde, Australia). The total surface area of capillary tubes formed was measured in five view fields per well using AxioVision 4.8 software and Matrigel™ Assay Recognition Software (M.A.R.S.) to quantitatively evaluate the vascular surface area of the structures formed.

Serial sections (10 μm) were fixed and stained using hematoxylin and eosin (H&E) immediately after sectioning and imaged using a bright field microscope (Zeiss Observer Z.1, Oberkochen, Germany) equipped with a Plan-APOCHROMAT lens and AxioCam MR3 camera, using a 20× or 40× magnification. High-resolution images of whole stained tissue sections were obtained using the stitching algorithm in Zeiss ZEN imaging software.

The antiangiogenic properties of treated or protein knockdown cells were determined by using the Matrigel™ assay. Briefly, 24-well plates were coated at 4 °C with 270 µL of Matrigel™ solution (1:1 dilution in cell culture medium) and were allowed to solidify at 37 °C for 1 h before seeding. HMEC-1 cells were then seeded at 1 × 10⁵ cells per well and allowed to adhere for 5 min before treatment was initiated, or without treatment for the knockdown cells. For the treatment, HMEC-1 cells were treated with 1 to 25 µg/mL of Dextran-Catechin or 20 µg/mL of TEPA. HMEC-1 cells were transfected with the optimized conditions for knockdown of CTR-1 (siRNA B, 20 nM) or ATOX-1 (siRNA C, 60 nM) for 6 hours and was incubated in fresh media for another 10 hours prior to the Matrigel™ assay. Photographs were taken after 8 hours using the 5X objective of an Axiovert 200 M fluorescent microscope coupled to an AxioCamMR3 camera driven by AxioVision 4.8 software (Carl Zeiss, North Ryde, Australia). The total surface area of capillary tubes formed was measured in 5 view fields per well using AxioVision 4.8 software and Matrigel™ Assay Recognition Software (M.A.R.S) to quantitatively evaluate the vascular surface area of the structures formed.