TTF1 Monoclonal Antibody (2F4D8)

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"Formalin-fixed paraffin sections (5µm in thickness) were cut. Sections underwent deparaffinization and rehydration. Endogenous peroxidase was blocked with methanol containing 3% hydrogen peroxide. Antigen retrieval was performed by microwaving the sections in citrate buffer, pH 6.0. The slides were allowed to cool at room temperature and washed 3 times by immersing them in TBST (Tris Buffered Saline having 0.05% Tween 20). Sections were incubated with the primary antibodies overnight at 4oC in humid chamber.
Next day, the slides were washed 4 times, 5 minutes each with TBST. Sections were then incubated for 30 minutes with the secondary biotinylated antibody followed by avidin peroxidase complex for another 30 minutes according to the manufacturer’s instructions (Universal Detection Kit, Dako, Denmark). The antigen was localized by the addition of DAB (Diaminobenzidine) substrate chromogen solution. Finally, slides were counterstained with hematoxylin, dehydrated in alcohol, and mounted.

For each setting, negative controls were carried out in which the primary antibody was omitted and replaced by PBS. Positive controls were squamous cell carcinoma of the lung for SOX2, red blood cells and perineurium of nerves (internal positive control) for GLUT-1, thyroid tissue for TTF-1 and lung tissue for RAGE."

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