Fluorescent microscope

One quarter of each retinal explant was carefully separated under microscope for subsequent immunohistochemical staining against the brain-specific homeobox/POU domain protein 3A (Brn3a). Unlike Thy1, which is another RGC-specific antigen, the expression pattern of Brn3a does not change after retinal injury and the levels of Brn3a protein are decreased with time after retinal injury, which is in agreement with the loss of RGC [151 (link)]. Therefore, Brn3a immunodetection is a powerful tool to assess RGC survival in rat injury models [152 (link),153 (link)]. Retinal tissue (n = 6 quarters/group) was fixed and stained as previously described [154 (link)]. Immunofluorescent RGCs were visualized and photographed with a fluorescent microscope (Carl Zeiss, Ltd., Hertfordshire, UK). Images of retinal whole mounts were photographed from 4 different regions of each quadrant of the retina (Figure 1) using a Zeiss fluorescent microscope (Carl Zeiss, Ltd., Hertfordshire, UK). Images were captured at a 20-fold magnification using a fluorescent camera (Carl Zeiss, Ltd., Hertfordshire, UK). Total numbers of Brn3a-positive cells were counted with the assistance of ImageJ (ImageJ Fiji v_1, total = 4 counts/quadrant, and six retinal quadrants per treatment group). The mean number of RGCs per quadrant was calculated from a mean count at each of the 4 different regions.

The cells of WM793 line were seeded on the glass microscopic slides in the 12-well plates. Double staining of cells with FITC-conjugated Annexin V and Propidium iodide (PI) (both from Sigma-Aldrich, St. Louis, USA) was used to identify early apoptotic events in human melanoma WM793 cells treated with Les-3833 and Dox (both used at 0.25 µg/mL concentration) for 72 h. After that, cells were washed with 1 × phosphate buffered saline (PBS), and incubated for 15 min with the Annexin V binding buffer containing 1/20 volume of FITC-conjugated Annexin V solution and PI (20.0 μg/mL) (14 (link)). The cells were analyzed under Zeiss fluorescent microscope (Carl Zeiss, Jena, Germany) using AxioImager A1 camera.
In 72 h after the addition of the tested compounds at 1 µg/mL concentration, the cells were stained with 0.2-0.5 µg/mL of the DNA-specific fluorescent dye Hoechst 33342 (Sigma-Aldrich, St. Louis, USA). The cells were also stained by the poly-specific dye Acridine orange (AO, 0.3-1.0 µg/mL, Sigma-Aldrich, St. Louis, USA). The cells were incubated for next 20-30 min and examined using a Zeiss fluorescent microscope (Carl Zeiss, Jena, Germany) using AxioImager A1 camera.

The formalin-fixed colonic sections were also subjected to TUNEL staining using a TUNEL assay kit (Roche Molecular Systems, MA, Switzerland). Nuclei were stained and images were acquired using a fluorescent microscope (Carl Zeiss AG, Jena, Germany). Hoechst 33,342 and images were acquired using a fluorescent microscope (Carl Zeiss AG, Jena, Germany). The number of apoptotic cells was counted as we described earlier [26 (link)].