Dp73 ccd digital camera

Four weeks after MSC transplantation, animals were sacrificed and the hearts were dissected. Frozen sections were prepared from each heart crossing the midlevel of the infarcted region. TUNEL assay was performed as mentioned above and cell nuclei were counterstained with hematoxylin. For each slide, color images of 10 randomly chosen high-power fields (HPF 200x) within the border zone were captured and digitized under a BX53 microscope with a DP73 CCD digital camera (Olympus, Tokyo, Japan). Images were analyzed by an investigator who was blinded with respect to the MSC treatment. TUNEL-positive cells were defined as cells with clear brown-colored nuclear labeling. On each image, the total area of the brown-colored nuclei (area_brown) and the area of total cell nuclei (area_total) were calculated. The apoptotic index was represented as the ratio of area_brown and area_total.

According to the standard histological techniques, the samples for microscopic examination were fixed for 24 h in 4% buffered paraformaldehyde, dehydrated with increasing concentrations of alcohol, embedded with paraffin, and cut into 6 µm-thick sections using microtome, Wetzlar, Germany. After removing the paraffin, sections were then stained with hematoxylin and eosin (HE) to determine the preservation of general histological structure and to evaluate the efficiency of cell debridement; additionally, the orcein stain was used to assess preservation of the vascular elastic elements. The specimens were examined under an Olympus IX83 light microscope. Images were collected and morphometric analysis was carried out using DP-73 digital CCD camera (Olympus, Tokyo, Japan) and CellSens Dimension (Olympus, Tokyo, Japan) image analysis system.

The testes were fixed in Carnoy’s solution overnight at 4 °C, dehydrated, embedded in paraffin, and sectioned at 7 µm with a Micro HM325 microtome (Thermo Fisher Scientific). Next, the sections were stained with Mayer’s hematoxylin and eosin (HE; Fujifilm Wako Pure Chemical, Osaka, Japan) according to a standard protocol. The frozen sections of testes were prepared as described above, permeabilized with 0.1% Triton X-100 in PBS for 10 min, blocked for 1 h in 10% Blocking One reagent in PBS at room temperature, and then incubated overnight at 4 °C with primary antibodies. The following day, the sections were washed three times in PBS and incubated with secondary antibodies for 1 h at room temperature. A DAB staining kit (Nacalai) was used to visualize USP26 and the nuclei were stained with 1 µg/ml DAPI. Immunofluorescence was observed using a BX51 fluorescence microscope (Olympus) and photographs were taken using a DP73 digital CCD camera (Olympus).

Usually, 20 Muscle-GO-GP prepupae were arranged in four groups of five samples on plastic Petri dishes with their dorsal side facing up. We recorded images at daily intervals using an Olympus MVX10 macrozoom microscope equipped with a DP73 digital CCD camera and cellSens acquisition software provided by the same manufacturer. Fields of view were recorded twice with filters for green and orange fluorescence, a zoom factor of 1.25, all of which resulted in digital colour images (TIFF or PNG format) of 2400×1800 pixels and a pixel size of 2.41μm per pixel. Phenotypes were assessed by visual inspection of individual images or montages.

Sperm isolated from the cauda epididymis were fixed in 1% paraformaldehyde (PFA) in phosphate-buffered saline (PBS) for 15 min at 4 °C, washed once in PBS, and then stained with 4 µg/ml biotin-labeled PNA (J–Chemical; Tokyo, Japan) and 1 µg/ml fluorescein isothiocyanate (FITC)-labeled streptavidin (Biolegend, San Diego, CA, USA) in PBS. Subsequently, the nuclei were stained with 1 µg/ml 4′,6-diamidino-2-phenylindole (DAPI).
The testes were fixed in 4% PFA in PBS overnight at 4 °C, washed twice in PBS for 1 h at 4 °C, and then rotated in 10% and 20% sucrose solutions in PBS overnight at 4 °C. The dissected testes were embedded in OCT compound, frozen, and sectioned at 7 µm with a HYRAX C50 cryostat (Carl Zeiss, Jena, Germany). The sections were permeabilized with 0.1% Triton X-100 in PBS for 10 min, blocked for 1 h in 10% Blocking One reagent (Nacalai, Kyoto, Japan) in PBS at room temperature, and then incubated for 1 h at 4 °C with 4 µg/ml biotin-labeled PNA and 1 µg/ml FITC-labeled streptavidin in PBS. Nuclei were stained with 1 µg/ml DAPI. The stages of the seminiferous tubules were determined based on acrosomal and nuclear morphology as previously described¹ . Finally, the sperm and the testis sections were analyzed under a BX51 fluorescence microscope (Olympus, Tokyo, Japan) and photographs were taken using a DP73 digital CCD camera (Olympus).