Ds fi1 ccd camera

Histology and immunohistochemistry were performed as previously described [22 (link)]. Briefly, wound tissue was fixed with 10% neutral-buffered formalin and paraffin sections (4 μm) stained with Mayer’s haematoxylin and eosin on randomly chosen samples. For immunohistochemistry, sections were incubated with rabbit anti-MMP-2 and anti-MMP-9 and isotyped-matched rabbit IgG (control; 1 µg/mL, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA). For immunodetection, a Dako EnVision+ System-HRP labelled polymer detection kit (Dako, Carpinteria, CA, USA) was used with ImmPACT NovaRED peroxidase (HRP) substrate (Vector Laboratories, Burlingame, CA, USA), and counterstained using Mayer’s hematoxylin and Scott’s bluing solution. After mounting, sections were observed under a light microscope (Eclipse Ci; Nikon, Tokyo, Japan) and micro-graphed using a DS-Fi1 CCD camera (Nikon, Tokyo, Japan). All samples were stained in a single assay to exclude between-run variability. The wound gap was measured by drawing a line the width of the wound, and the number of pixels was recorded using ImageJ. The pixels of the line were divided by the pixels of the scale bar as indicated in the figures.

The spheres were transferred into a cell-culture chamber slide (Labtek II; Nunc, Waltham, MA, USA) and incubated in f-EM, under adhesion-condition culture at 37 °C in 5 % CO₂ to allow their attachment. After 2 hours, cells were washed with phosphate-buffered saline (PBS) and fixed for 30 minutes in 2 % (wt/vol.) paraformaldehyde in PBS at room temperature, then incubated with 1 μg/ml PE-conjugated anti-human SSEA-4 monoclonal antibody (Miltenyi Biotec, Bergisch Gladbach, Germany) in PBS/bovine serum albumin (BSA; PAA) for 30 minutes, at room temperature. After incubation the cells were washed three times with PBS, counterstained with DAPI (Sigma Aldrich), and observed under a Zeiss Axiophot fluorescence microscope equipped with a Nikon DS-FI1 CCD camera.
Human bone marrow-derived MSCs (BM-MSCs; Lonza, Walkersville, MD, USA) were used as a stem marker positive control, HeLa (kindly obtained from Dr Salvatore Feo at University of Palermo) were used as an LESC marker positive control, and unstained f-LSCs were used as a negative control.

Observations were made under an Eclipse E-600 epifluorescence microscope (Nikon, Japan) using B2 filter (blue light; λ = 465–496 nm) for the rabbit anti-chicken FITC-conjugated antibody and DiOC6(3), and U2 filter (UVB light; λ = 340–380 nm) for DAPI- and AB-stained cells. All images were recorded at exactly the same time of integration using DS-Fi1 CCD camera (Nikon, Japan). Axial scans taken along representative cell files were plotted using ImageJ software. Confocal observations were made with Leica SP8 (Germany) using laser lines 405 nm (DAPI) and 553 nm (Alexa Fluor^® 555). Images were acquired with HCPL APO 63×/1.40 oil. Confocal microscopy was performed in the Laboratory of Microscopic Imaging and Specialized Biological Techniques at the Faculty of Biology and Environmental Protection, University of Lodz. The cell cycle positions (if needed) were estimated by morphometric analysis (cell length measurements) combined with the cytometric analysis of nuclear DNA (C value) after DAPI staining. All immunofluorescence analyses and Western blot assays were repeated at least twice, while other experiments were repeated several times. For statistical analysis, the intensities of immunofluorescence were measured for 50 cell wall areas in 50 (2 cell stage) and 25 (later stages) individual filaments. One-way ANOVA and Bonferroni post hoc test were applied.