Immunocytochemical localization of intermediate filaments cytokeratin and vimentin
This protocol is extracted from research article:
Reversible EMT and MET mediate amnion remodeling during pregnancy and labor
Sci Signal, Feb 11, 2020; DOI: 10.1126/scisignal.aay1486

Immunocytochemical staining for vimentin (3.7 μl/ml; Abcam, ab92547) and CK-18 (1 μl/ml; Abcam, ab668) were performed for multiple experimental end points (13). In addition, vimentin and CK-18 were measured during PGRMC2 overexpression studies. The manufacturer’s instructions were used to calculate staining dilutions to ensure uniform staining. After each time point, cells were fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X-100, and blocked with 3% BSA in PBS before incubation with primary antibodies overnight at 4°C (38). This protocol is adequate to remove nonspecific binding of primary antibodies in our system. After washing with PBS, slides were incubated with Alexa Fluor 488– and Alexa Fluor 594–conjugated secondary antibodies (Life Technologies), diluted 1:1000 in PBS, for 1 hour in the dark. Slides were washed with PBS, treated with NucBlue Live ReadyProbes Reagent (Life Technologies), and then mounted using Mowiol 4-88 mounting medium (Sigma-Aldrich).

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