Fresh frozen cancerous melanoma tissues sections (6–8 mm) were cut under tissue freezing media (Bio-Optika, Milano, Italy) by Slee Cryostat mnt (Auroscience), and were fixed by 4% paraformaldehyde (PFA) PBS for 15 minutes. Chamber slide (LAB-TEK Brand Products, Nalgen Nunc, Naperville, US) cultures were blocked with 3% BSA PBS, before tumor-associated disialylated glycosphingolipid-specific antibodies and the selected antibody fragments were added and reacted at 4°C overnight. Immunofluorescence labelling using second antibodies, was detected in confocal laser microscopy (Nikon Eclipse E600, Nikon Model C1-Lu3, Tokyo, Japan) or conventional IF microscopy.
Model c1 lu3
Manufactured by Nikon
Sourced in Japan
The Nikon Model C1-Lu3 is a laboratory equipment designed for microscopy applications. It is a confocal laser scanning microscope system that provides high-resolution imaging capabilities. The core function of the Nikon Model C1-Lu3 is to enable researchers and scientists to capture detailed and high-quality images of specimens under examination.
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2 protocols using model c1 lu3
1
Immunofluorescence Analysis of Melanoma
2
Immunofluorescent Labeling of Melanoma Tissue
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Minor melanoma tissue samples were snap-frozen with isopentane in liquid nitrogen. Fresh frozen cancerous tissue cryostat sections [6–8 μm, freezing media (Bio-Optika, Milano, Slee Cryostat mnt (Auroscience)] of melanomas were fixed in 4% paraformaldehyde (PFA) PBS for 15 min. Three percent BSA PBS was used for blocking, before the monoclonal antibodies specific to tumor-associated disialylated glycosphingolipid antigens (Calbiochem, Abcam) were added for an overnight incubation at 4°C. Indirect immunofluorescence with FITC-labeled second antibodies (anti mouse IgG FITC from DAKO), or biotinylated rabbit anti mouse IgG (Fab')2 (1:100) and Streptavidin FITC (Vector Laboratories, Burlingame, CA, USA) with propidium-iodide (nuclear staining) was used. Chamber slides (Nunc Lab Tech) were used in certain cases to culture cancerous cells and IF label them in situ. Indirect immunofluorescence (FITC) labeling was detected in confocal laser microscopy (Nikon Eclipse E600, Nikon Model C1-Lu3, Tokyo, Japan) or conventional IF microscopy.
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