Monochrome ccd camera

Immunocytochemistry assessments were accomplished as described previously.²³, ⁴⁵ Briefly, cells of WiDr or SW48‐Dox lines were pretreated with Genz‐161 or transfected with siRNA‐Gb3 synthase (100 nM) for 48 hours, then placed (8000/chamber) in four‐chamber slides and cotreated with 100 nM doxorubicin for 48 hours in 5% FBS medium. After wash and fixation, cells were blocked with 5% goat serum in PBS for 30 minutes at room temperature, and then incubated with primary antibodies, anti‐p21 (1:200), ‐phosphorylated p53 (pp53), and ‐METTL3 (1:1500) in blocking solution, at 4ºC overnight. Corresponding Alexa Fluor 488‐conjugated anti‐mouse IgG and Alexa Fluor 555‐conjugated anti‐rabbit IgG (1:1000) were applied for further incubation to recognize the corresponding primary antibodies. After washing, cell nuclei were counterstained with DAPI (4̍,6‐diamidino‐2‐phenylindole) in mounting solution (Vector laboratories, Burlingame, CA, USA). Images (200 × magnification) were captured with an Olympus BX63 automated fluorescence microscope with monochrome CCD camera (Olympus, Tokyo, Japan). Alexa Fluor 488‐conjugated anti‐mouse IgG and Alexa Fluor 555‐conjugated anti‐rabbit IgG were purchased from Thermo Fisher Scientific.

A stereoscopic microscope equipped with a monochrome CCD camera (Olympus Co., Japan) was used for image observation of the mycelia. The captured images were fed into a computer and analyzed using the image analysis software (Patent ID: 2011R11S044664.). The captured images were thresholded to obtain binary images. Opening was then applied to the binary images to improve their qualities. Repeated opening cycles were applied to each pellet until only the pellet core remained. Subtraction of the pellet core from the whole mycelia provided the filamentous part. For each sample, images of at least 20 colonies (pellet + filaments) were used to determine the morphological parameters. The average value was used to analyze the morphological parameters. The projected area of the whole mycelia (A_m), area of the pellet core (A_pc),equivalent diameters of the projected area of the whole pellet (D_m) and the diameter of pellet core were determined by the method developed by Koike et al. [26 ]. The fluffy degree of the pellet was given by A_pc/A_m. Fifty elements (defined as either pellets or dispersed filaments) were randomly selected in shake flasks, and then dried at 80°C to get the dry cell weight (DCW). We ideally regarded the mycelia as a regular sphere to obtain its volume. The compactness was determined as follows:
$\mathrm{c}\mathrm{o}\mathrm{m}\mathrm{p}\mathrm{a}\mathrm{c}\mathrm{t}\mathrm{n}\mathrm{e}\mathrm{s}\mathrm{s}=\frac{\mathrm{D}\mathrm{C}\mathrm{W}/50}{\frac{4}{3}\times \mathrm{\pi }\times {\left(\frac{{\mathrm{D}}_{\mathrm{m}}}{2}\right)}^3}$