Lsm700 confocal microscopy system

The number of apoptotic cells was analyzed by TdT-mediated dUTP nick end labeling (TUNEL), using the in situ Cell Death Detection Kit, Fluorescein (Roche 11684795910) following the manufacturer's directions. Twelve-micrometer cryosections were collected on slides and subjected to TUNEL assay. To consider the presence of unspecific results, some retina sections were incubated with the reaction mix without TUNEL reaction enzyme. Sections were observed with a Leica DM-5500 microscope and then confocal images were acquired using the LSM700 Zeiss Confocal Microscopy system. The number of TUNEL-positive cells was evaluated in the dorsal and ventral part of the retina by manual counts with a Leica DM-6000 microscope, with the objective Leica ∞/0.17/D, HCX PL FLUOTAR, 40X/0.75 that has an area of 0.31 mm².

Fluorescent images of dorsal and ventral region of mice retina were captured at 20X magnification using LSM700 Zeiss Confocal Microscopy system, converted to gray-scale and normalized to background staining, using ImageJ. Quantification of GFAP+ reactivity was measured as mean values to define fluorescence signal intensity (IntDen/Area) and as the area occupied by fluorescent-labeling in each region of interest.
To quantify the numbers of microglial cells, the number of Iba-1-positive cells was evaluated in the dorsal and ventral part of the retina by manual counts in each considered retinal layer: GCL, IPL, OPL, and ONL. These counts were pooled to obtain a mean number of microglial cells per layer, per retinal region and per animal group.

Tumor tissues were isolated and frozen in O.C.T. (Sakura Finetek; Yokohama, Kanagawa, Japan), and cryosections (8 μm) were sliced. Cryosections or ovarian cancer cells were prefixed in 4% paraformaldehyde for 20 min at room temperature. After permeabilization with 0.5% Triton X-100 (Sigma-Aldrich; St. Louis, MO, USA), 5% donkey serum was used for blocking for 1 h at room temperature. Immunostaining was performed using primary antibodies overnight at 4 °C, and samples were subsequently treated with Alexa Fluor-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories; West Grove, PA, USA) for 1 h at room temperature. Nuclei were counterstained with Hoechst 33,258 (Sigma-Aldrich, 14,530). Images were acquired using a Zeiss LSM 700 confocal microscopy system (Carl Zeiss Jena, Oberkochen, Germany) and quantified with ImageJ software. Skeletonization of CD31 staining was performed and vessel length was measured as previously described [25 (link)].