Eclipse e600 microscope

Cells and blastoids were fixed in 4% paraformaldehyde for 20 min, rinsed in PBS, permeabilized with 0.5% Triton X-100 (Sigma-Aldrich, Milan, Italy) for 30 min, and incubated with a blocking solution containing 10% goat serum (Sigma-Aldrich, Milan, Italy) for 30 min. ICM-like spheroids were dissociated and attached to slides using a cytocentrifuge, Cytospin 4 (Thermo Shandon, Milan, Italy), before immunocytochemical staining. Primary antibodies for OCT4 (1:200, Chemicon, Milan, Italy, ab3209), KRT19 (1:200, Abcam, Cambridge, UK, ab76539), and CDX2 (1:50, Santa Cruz Biotechnology, Milan, Italy, sc-166830) were incubated overnight at +4 °C. Samples were then washed with PBS and incubated with the appropriate secondary antibodies (Alexa Fluor) for 45 min at room temperature using a 1:250 dilution. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich, Milan, Italy). At the end of the immunostaining procedure, cells were analyzed under an Eclipse E600 microscope (Nikon, Firenze, Italy) equipped with a digital camera (Nikon, Firenze, Italy); blastoids were transferred, mounted to glass slides, and visualized under an Eclipse E600 microscope (Nikon, Firenze, Italy) equipped with a digital camera (Nikon, Firenze, italy). Images were acquired using NIS-Elements Software (Version 4.6; Nikon).

For Cox-2, immunolabeling was quantified using a semi-quantitative method adapted from [36 (link)], based on the percentage of positive tumor cells (extension) and the intensity of staining. The percentage of the positive cells was given scores ranging from 1 to 3 (1 for ≤10%, 2 for 11–50%, 3 for >51%), while the intensity of staining was also scored from 1 to 3 (weak, moderate, and strong). These scores were combined to produce a final score, calculated as the product of extension and intensity, categorizing the samples as Low (score < 6), and high expression (score ≥ 6).
The immunoreactivity of the EGFR antibody was considered positive when membranous staining above the background level in greater than 1% of tumor cells was detected. The intensity of the staining was evaluated as previously described. High expression was considered in cases where the staining of the membrane was of strong intensity [37 (link)].
All samples were independently evaluated by two observers (IP and JP), who were blinded to clinical and pathological characteristics, using a Nikon Eclipse E600 microscope coupled with a Nikon DXM1200 digital camera, provided by Nikon Instruments Inc., Melville, NY, USA. A third reviewer (LD) was consulted in cases of inconsistent findings. A consensus discussion was then held to determine the final score.