Dapi nuclear stain

Subgroups of hearts were harvested at 4 and 14 weeks after TAC for analyses of structure, fibrosis, and biochemistry. Trichrome staining was performed as previously described (44 (link)). Briefly, mice were euthanized, and after cardioperfusion, hearts were fixed for 1 to 3 days in 4% paraformaldehyde at 4°C. Hearts were dehydrated and paraffinized using the Microm STP 120 from Thermo Fisher Scientific, embedded in paraffin using a HistoStar apparatus (Thermo Fisher), and sectioned (4 to 6 μm) using the Microm HM 325 (Thermo Fisher). Tissue sections were then stained with Weigert’s iron hematoxylin and Masson trichrome (Sigma-Aldrich) according to the manufacturer’s instructions. Interstitial fibrosis was quantified by color threshold measures using ImageJ. Alternatively, tissue sections were deparaffinized and rehydrated according to the trichrome staining protocol, but after the wash with deionized water, heart sections were washed three times for 5 min with 1× phosphate-buffered saline (PBS) followed by staining with Alexa Fluor 488–conjugated WGA (1:10 in 1× PBS) (Invitrogen) for 1 hour at room temperature in a humidified chamber in the dark. Sections were again washed three times for 5 min with 1× PBS followed by mounting with coverslips using Fluoromount-G mounting media containing DAPI nuclear stain (Southern Biotech).

Hearts from 8-week TAC studies were harvested for analyses of CSA and fibrosis. Masson’s trichrome staining was performed as previously described(45 (link)). Briefly, mice were euthanized, and hearts were fixed overnight in 4% paraformaldehyde at 4°C. Hearts were dehydrated and paraffinized followed by embedding in paraffin blocks. Blocks were sectioned at 5 μm and stained with Weigert’s iron hematoxylin and Masson trichrome (Sigma-Aldrich) according to the manufacturer’s instructions. Alternatively, tissue sections were deparaffinized and rehydrated according to the trichrome staining protocol, but after the wash with deionized water, heart sections were stained with Alexa Fluor 594–conjugated wheat germ agglutinin (10μg/mL in 1× PBS) (Invitrogen) for 1 hr at room temperature in a humidified chamber in the dark. Sections were washed 3 times for 5 min with PBS followed by mounting with coverslips using Fluoromount-G mounting media containing DAPI nuclear stain (Southern Biotech). CSA was measured at 20x magnification by tracing individual cells in ImageJ. Fibrosis was assessed by a thresholding method in ImageJ as described previously(46 ).

To evaluate the expression and distribution of E-cadherin in PDACs after regulated ESE3 expression, PDAC cells were seeded onto glass slides, washed with phosphate-buffered saline, fixed with 4% paraformaldehyde for 15 minutes, permeabilized with 0.1% Triton X-100 in phosphate-buffered saline for 15 minutes at room temperature, and blocked for 1 hour with 3% bovine serum albumin in phosphate-buffered saline. The cells were then stained with an anti-E-cadherin antibody (1:200 dilution) overnight at 4°C. Cells were mounted with 4’,6-diamidino-2-phenylindole (DAPI) Fluoromount-G medium and a DAPI nuclear stain (SouthernBiotech). The slides were viewed under an Olympus microscope.