Histobond adhesive microscope slide

First, 2 × 10⁷ coumarin-labeled MB was incubated with the adhered J774A.1 cells for 1, 5, and 10 min at 37 °C. Upon incubation, the samples were then washed with PBS, fixed with 4% paraformaldehyde for 10 min, and again washed with PBS at room temperature. To stain the cell membrane and nucleus, samples were incubated with wheat germ agglutinin (1:500) Alexa Fluor 594 (ThermoFisher Scientific, Massachusetts, USA) and DAPI (1:500) (ThermoFisher Scientific, Massachusetts, USA), respectively, at room temperature for 30 min. Subsequently, the samples were washed with PBS several times, and the 12-mm round coverslips used as cell supports were removed from the 24-well plates using surgical tweezers. Finally, a drop of Mowiol mounting medium was placed on each HistoBond adhesive microscope slide (Paul Marienfeld GmbH & Co.KG, Lauda-Königshofen, Germany) for adhering and attaching the coverslips.

To confirm the shape of the formed MB, we employed an Olympus BX41 microscope equipped with a microEye camera (Carl Zeiss, Oberkochen, Germany). We placed a drop of the sample on a HistoBond adhesive microscope slide (Paul Marienfeld GmbH & Co.KG, Lauda-Königshofen, Germany) and measured it in bright-field mode. The images were acquired using Olympus 50× LMPlanFL N objective. MB size was measured from bright-field images using ImageJ software. The aspect ratio and surface area were estimated from the two-dimensional (2D) projection images and mathematical equations for the respective shapes.