Flouview fv 1000 mpe

FJB staining was performed as previously described [13 (link)] with some modifications. After air-drying the tissue slides overnight, the slides were immersed in a solution of 1% sodium hydroxide and 80% ethanol for 5 min. Then, slides were immersed in 70% alcohol and distilled water for 2 min each. Tissue slides were transferred to a solution of 0.06% potassium permanganate for 10 min, rinsed with distilled water and then immersed in a solution of 0.1% acetic acid and 0.01% FJB for 20 min. The slides were washed with distilled water and allowed to dry for 10 min. Glass coverslips were mounted using Dibutylphthalate Polystyrene Xylene (DPX) non-fluorescent mounting medium, and images were assessed with a confocal laser scanning microscope (Flouview FV 1000 MPE, Olympus, Tokyo, Japan). The images were proceeded for quantification using the computer-based ImageJ program.

An immunofluorescence study was carried out. Slides with brain tissue samples were washed twice for 10 min in 0.01 M PBS (Phosphate-Buffered Saline) by adding a proteinase K solution. Tissue samples were incubated for 60 min in blocking solution covering 2% normal goat/mouse serum and 0.3% Triton X-100 in PBS. Slides were further incubated overnight in primary antibodies at 4 °C, and further incubated at room temperature for 2 h in secondary antibodies (goat anti-mouse, fluorescein isothiocyanate (FITC)-labeled secondary antibodies (1:50 in PBS) Santa Cruz Biotechnology), after incubating the primary antibodies. The mounting medium was applied to cover the slides with glass coverslips after counterstaining with DAPI (4,6-diamidino-2-phenylindole) (for 10 min). Immunofluorescence was studied using a confocal laser-scanning microscope (Flouview FV 1000MPE, Olympus, Japan); for immunohistological quantitative analysis, ImageJ software was used (v. 1.50).

Immunofluorescence staining was performed as previously described, with a modification [42 (link),43 (link)]. Initially, the slides were dried overnight before staining, followed by washing with PBS (0.01 Mm), twice, for 10 min. The slides were incubated with proteinase K for 5 min and then rinsed with PBS (0.01Mm). For blocking, 2% normal serum was applied (goat/rabbit) with 0.1% Triton X-100 in PBS. After blocking, the slides were carefully incubated with primary antibodies, overnight, including Glial fibrillary acidic protein (GFAP), Caspase-3, p-JNK, IL-1β Iba-1, PSD-95, and Synaptophysin. The secondary antibodies, Tetramethylrhodamine (TRITC) and Fluorescein isothiocyanate (FITC)-labeled (1:100), were incubated for 90 min, followed by 4′,6-diamidino-2-phenylindole (DAPI) for the nucleus detection. The slides were covered using coverslips with a mounting medium, and images were taken through a confocal laser-scanning microscope (Flouview FV 1000 MPE, Olympus, Japan ). For the quantification of the amount of staining in the fluorescence images obtained with confocal microscopy, Integrated Density was used. Integrated Density was determined using ImageJ software (version 1.50, NIH, https://imagej.nih.gov/ij/, USA) and it represents the sum of the values of the pixels in an image.