Hoeschst 33342

The antitumor effect of BS was assessed in MCF-7 cells using the dyes fluorescein diacetate (FDA, 7.5 µg/mL), Hoeschst 33.342 (Ho, 4.0 µg/mL), and propidium iodide (PI, 1.0 µg/mL) (Sigma-Aldrich, USA) for analysis of apoptosis or necrosis, through the presence or absence of apoptotic bodies, respectively^{20 (link)}. Cells were exposed to BS (1–10 µg/mL) for 72 h with subsequent staining with fluorochromes and counting 300 cells/treatment. Phosphate buffered saline and Docetaxel (DT 50 µM) were used as negative (NC) and positive (PC) controls, respectively. The cells were analyzed by microscopy (Olympus BX 43, Olympus Microscopy, Europe) to assess the percentage of viable cells (FDA+/Ho+); in early (FDA+/Ho+), late (FDA+/Ho+/PI+) apoptosis; and necrosis (FDA−/Ho−/PI+)^{21 (link)}.

Sections were first air-dried for 1 h at RT. After several rinses with PB, the sections were pre-treated for 15 min with 10% methanol in PB and washed three times with PBS for 10 min each time. In the case of myelin basic protein (MBP) labelling, sections were delipidated with a battery of ethanol-increasing concentrations (25%, 75%, 95%, and 100%) and later step-back rehydration for the following steps. Sections were then pre-incubated for 1 h at RT in incubation buffer: 5% normal donkey serum (EMD Millipore, Billerica, MA, USA) and 0.2% Triton X-100 (Sigma-Aldrich, Madrid, Spain) diluted in PBS. Immunohistochemistry was performed by incubating the sections overnight at 4 °C with the primary antibodies (Table 2) diluted in incubation buffer. After rinsing, the sections were incubated with the corresponding fluorescent (1:1000, Invitrogen, Paisley, UK) secondary antibodies in the incubation buffer for 1 h at RT. In all cases, cell nuclei were stained with Hoeschst 33342 (10 µg/mL, Sigma-Aldrich, Madrid, Spain) and the sections were mounted with coverslips in Fluoromount-G (Southern Biotech, Birmingham, AL, USA).

Fibroblasts were cultured on coverslips or 96-well plates (Corning) for 24–72 h in DMEM media. Cells were treated according to the rescue protocol or left untreated. Cells were fixed with 3.7% formaldehyde at room temperature for 15 min, washed three times with PBS, and permeabilized for 15 min with 0.2% Triton X-100 (Sigma) in PBS. Cells were washed three times with PBS and blocked with 1% bovine serum albumin (Invitrogen) for 15 min. Cells were incubated for one hour at room temperature with anti-CNN2 (Sigma; HPA049095) and anti-FLAG (Cell Signaling; 8146) primary antibodies diluted 1/1000 in a TX-100/BSA buffer containing 0.2% BSA and 0.004% TX-100 in PBS. Cells were washed in PBS and incubated with AlexaFluor-488 anti-rabbit or AlexaFluor-594 anti-mouse antibodies for one hour, covered at room temperature. Cells were washed with PBS and stained with 1ug/mL Hoeschst33342 (Sigma). Cells were imaged using the Olympus Fluoview FV-1000 Laser Confocal Microscope.

For the cell cycle analysis, 1.5 × 10⁶ purified CD34⁺ cells from cord blood were suspended in Hoeschst Binding Buffer (Hank’s Balanced Salt Solution, containing 20 mM HEPES, 5.5 mM glucose and 10% HyClone) and stained for DNA and RNA with Hoeschst 33342 (Sigma-Aldrich, St. Louis, Missouri, USA) for 45 min at 37 °C in darkness. Then, pyronin Y (#P9172-IG, Sigma-Aldrich) was added and cells were incubated in the same conditions. Finally, cells were surfaced stained with hCD34 (#555824, BD), hCD38 (#303522, BioLegend), and hCD18 (#555923, BD Pharmingen) monoclonal antibodies and were analyzed by flow cytometry.

INS-1 cells and pancreatic sections were rinsed twice in PBS and then incubated with specific primary antibodies overnight at 4 °C. The following antibodies were used: rabbit anti-LC3 (1 : 200; CST), mouse anti-LAMP2 (1 : 200; MBL, Nagoya, Japan), guinea pig anti-insulin (1 : 200; Abcam, San Francisco, CA, USA) and mouse anti-glucagon (1 : 200; CST). The next day, INS-1 cells and pancreatic sections were incubated with secondary antibodies (Invitrogen) using donkey anti-mouse IgG (Alexa Fluor 488; 1 : 500), donkey anti-guinea pig IgG (Alexa Fluor 594; 1 : 500) and goat anti-rabbit IgG (Alexa Fluor 488; Alexa Fluor 594; 1 : 500). Subsequently, the samples were stained with Hoeschst 33342 (Sigma-Aldrich) diluted 1 : 1500 in PBS. Stained cells and tissues were viewed using an Olympus FV1000 confocal laser-scanning microscope with excitation filters at 488 nm (green) and 594 nm (red).

For immunocytochemical analysis, the cells at 3rd or 4th passage were seeded on 24-well plates (Nunc, Thermo Fischer Scientific) at 2.5 × 10³ cells/cm² density. At 70% confluence, the cells were washed carefully in PBS (Macopharma), fixed with 4% PFA (Sigma-Aldrich) for 15 min at room temperature and washed in PBS (Sigma-Aldrich) again. An amount 0.2% Triton X-100 (Sigma-Aldrich) was used to permeabilize cell membranes in case of detecting intracellular target antigen. To block nonspecific binding, a mixture of 10% goat serum (Gibco) and 1% bovine serum albumin (Sigma-Aldrich) was applied for one hour. Subsequently, cultures were washed with PBS (Sigma-Aldrich) and incubated with primary antibodies (Table 2) for 24 h at 4 °C. For every variant of staining, negative control was performed to analyze the specificity of the reaction. On the following day, the cells were washed in PBS (Sigma-Aldrich), and the secondary antibodies (Table 3) were added in darkness for one hour. After the cells were washed with PBS (Sigma-Aldrich) again, the nuclei were stained with Hoeschst 33342 (Sigma-Aldrich) for 15 min.
The samples were analyzed with LSM 780 confocal laser scanning system and ZEN software (Carl Zeiss). Quantitative analysis was performed as a relation of positive cells to all cells (50 cells in one repetition). Each variant had 3 repetitions.