Fluorescence in situ hybridization (FISH) of cultured cells and tissue monolayers was performed as previously described.20 (link) Briefly, cells were fixed and immersed in 70% ethanol at 4°C overnight. After treatment with wash buffer and prehybridization buffer, slides were incubated with (CAG)6CA-5′ Texas red-labeled 2-O-methyl RNA 20-mers probe (IDT). The next day, slides were washed twice and then stained with mounting media with DAPI (H-1500; Vector Labs, Burlingame, CA, USA). For detection of MBNL protein, prior to ethanol treatment, permeabilization with 0.2% Triton 100 in 2× SCC was performed. After probe incubation, cells were incubated with anti-MBNL1 or anti-MBNL2 antibody.
The slides were sealed and imaged at 60× magnification using a Widefield Deltavision microscope. Images were processed by deconvolution with AutoQuant X3 (Media Cybernetics, Rockville, MD, USA) software. Visualization of RNA foci and co-localization of MBNLs proteins were made using ImageJ (National Institutes of Health, Bethesda, MD, USA). For quantification, data were analyzed from at least 10 pictures containing greater than 50 cells each. For cells, data were analyzed from two batches of F35T cells. For tissues, data were analyzed from five FECD tissues.
The slides were sealed and imaged at 60× magnification using a Widefield Deltavision microscope. Images were processed by deconvolution with AutoQuant X3 (Media Cybernetics, Rockville, MD, USA) software. Visualization of RNA foci and co-localization of MBNLs proteins were made using ImageJ (National Institutes of Health, Bethesda, MD, USA). For quantification, data were analyzed from at least 10 pictures containing greater than 50 cells each. For cells, data were analyzed from two batches of F35T cells. For tissues, data were analyzed from five FECD tissues.
