Eclipse te2000 s inverted microscope

Cells (75,000) were seeded into 6-well plates. Unless otherwise stated, the medium was replaced the following day with either normal DMEM medium, low-serum DMEM (1% FBS), or drug-/peptide-supplemented DMEM. Then, after overnight incubation, images were taken with a Nikon Eclipse TE2000-S inverted microscope at 10× magnification. Between 5 and 10 images were obtained for each condition in each independent experiment, and the numbers of cells and TNTs were counted to calculate the ratio of TNTs/cell.

Expression of CDCP1 was analyzed by IHC on consecutive 2-μm formalin-fixed, paraffin-embedded (FFPE) tumor sections, using rabbit polyclonal anti-CDCP1 (1:50) (PA5-17245, Thermo Fisher Scientific), which is directed against the C-terminus of CDCP1, after antigen retrieval, which as performed by heating the sections for 5 min at 96°C in 10 mM citrate buffer, pH 6.0. Immunoreactions were visualized using streptavidin-biotin-peroxidase (Dako, Agilent Technology, Santa Clara, CA), 3,3′-diaminobenzidine (DAB; brown signal) (Dako) as the chromogen, and the sections were counterstained with hematoxylin. Images were acquired on an ECLIPSE TE2000-S inverted microscope (Nikon Instruments, Melville, NY) at 20× and 40× magnification.
The reactivity of anti-CDCP1 in the TNBC specimens was considered to be positive when ≥ 10% of tumor cells showed membrane staining. This cutoff was chosen, based on distribution analysis of the percentage of CDCP1-positive cells by IHC in each tumor section. No tumors had < 10% CDCP1-positive cells in our series, and cases with different percentages of CDCP1-positive cells were likewise distributed in a 10–100% interval. By explorative Kaplan-Meyer analysis of DFS in our cases—stratified as negative, ≥ 10% and < 50%, or ≥ 50% for CDCP1 expression, both CDCP1-positive groups had a worse and superimposable DFS compared with CDCP1-negative cases (data not shown).

Microscopic analyses were performed using epifluorescence DM2000 LED microscope (Leica) equipped with a DF450 C camera (Leica) or an Eclipse TE2000-S inverted microscope (Nikon) equipped with a DC 300F camera (Leica). Images were acquired using the Leica Application Suite 4.2 software (Leica).

Multilineage differentiation potential of UC-MSC was assessed using StemPro Adipogenesis (A10070-01, Gibco), Osteogenesis (A10072-01, Gibco) or Chondrogenesis (A10071-01, Gibco) kits, as per manufacturer instructions. After differentiation, adipogenesis induction was assessed by Sudan Black staining. Briefly, cells were fixed with 4% paraformaldehyde solution for 45 min at room temperature, stained with a Sudan Black saturated solution in 70% ethanol for 5 min at room temperature and finally washed thoroughly with 70% ethanol. Osteogenesis induction was assessed by Alzarin Red staining. Fixed cells were stained in a 2% Alzarin Red solution (pH 4.2) for 3 min at room temperature and then washed thoroughly with distilled water. Finally, chondrogenic induction was assessed by Alcian Blue staining. Fixed cells were stained in a 1% Alcian Blue solution prepared in 0.1 N HCl for 30 min at room temperature and then washed thoroughly with distilled water. All three preparations were visualized under light microscope. Images were acquired using a Nikon Eclipse TE2000-S inverted microscope and the Eclipse Net software.

hPSCs were analyzed for in situ immunofluorescence^{5 (link),56 (link)}. Briefly, cells were rinsed with ice-cold PBS and fixed in PBSA (PBS with 0.1% bovine serum albumin) with 4% formaldehyde for 45 min. After two washes cells were permeabilized with 0.1% Triton X-100 in PBSA with 10% normal goat serum for 30 min, washed twice, and stained with a rabbit polyclonal antibody anti-active CASPASE-3 (ab13847, Abcam Inc., Cambridge, MA, USA). Fluorescent secondary antibody Alexa Fluor 488-conjugated anti-rabbit IgG (Thermo Scientific) was used to localize the antigen/primary antibody complexes. Cells were counterstained with DAPI and examined under a Nikon Eclipse TE2000-S inverted microscope equipped with a 20X E-Plan objective and a super high-pressure mercury lamp. The images were acquired with a Nikon DXN1200F digital camera controlled by the EclipseNet software (version 1.20.0 build 61).

HPAECs were grown in 35 mm glass bottom dishes and treated with control conditions, hemin, or hemin and ML-7 for 24 h. Cells were fixed in 3.7% paraformaldehyde, permeabilized with 0.25% fish skin gelatin, 0.01% saponin, 0.1% NaN₃ in PBS, and then incubated in primary antibodies overnight. Cells were subsequently incubated with appropriate fluorochrome-conjugated secondary antibodies for 1 h. Analysis and photos were obtained using Nikon Eclipse TE2000-s inverted microscope.