Ma5 15065

VEH- and EDC-MMSCs were seeded onto coated-glass coverslips and culture under the conditions stated above. Confluent cells were then fixed with 4% paraformaldehyde for 15 minutes, followed by permeabilization with 0.1% Triton X-100 in PBS for 15 minutes at room temperature. Non-specific binding was blocked with 2% BSA in PBS for 1 hour at room temperature. Primary antibody targeting TGFβ1 (MA5–15065, Invitrogen, 1:100 in 0.1% BSA-PBS) and TGFβ Receptor I (PA-95863, Invitrogen, 1:100 in 0.1% BSA-PBS) were applied and incubated overnight at 4°C. The cells were then washed with PBS three times for 5 min and incubated with Alexa Fluor^™ 568-conjugated α-Rabbit secondary antibody (A11011, Invitrogen, 1:2000 in 0.1% BSA-PBS) for 1 hour at room temperature. Finally, the coverslips were mounted onto glass slides using mounting medium containing DAPI for nuclear counterstaining (H-1200, VECTASHIELD). Digital image files were created with an Olympus VS200 Research Slide Scanner (Olympus / Evident, Center Valley, PA) with a Hamamatsu ORca-Fusion camera (Hamamatsu Photonics, Skokie, IL). Individual images were created with the OlyVIA Viewer software (Olympus / Evident, Center Valley, PA). Negative controls without primary antibody were included to validate the staining specificity.

VEH- and EDC-MMSCs were seeded onto coated-glass coverslips and culture under the conditions stated above. Confluent cells were then fixed with 4% paraformaldehyde for 15 min, followed by permeabilization with 0.1% Triton X-100 in PBS for 15 min at room temperature. Non-specific binding was blocked with 2% BSA in PBS for 1 h at room temperature. Primary antibody targeting TGFβ1 (MA5-15065, Invitrogen, 1:100 in 0.1% BSA-PBS) and TGFβ Receptor I (PA-95863, Invitrogen, 1:100 in 0.1% BSA-PBS) were applied and incubated overnight at 4 °C. The cells were then washed with PBS three times for 5 min and incubated with Alexa Fluor™ 568-conjugated α-Rabbit secondary antibody (A11011, Invitrogen, 1:2000 in 0.1% BSA-PBS) for 1 h at room temperature. Finally, the coverslips were mounted onto glass slides using mounting medium containing DAPI for nuclear counterstaining (H-1200, VECTASHIELD). Digital image files were created with an Olympus VS200 Research Slide Scanner (Olympus/Evident, Center Valley, PA) with a Hamamatsu ORca-Fusion camera (Hamamatsu Photonics, Skokie, IL). Individual images were created with the OlyVIA Viewer software (Olympus/Evident, Center Valley, PA). Negative controls without primary antibody were included to validate the staining specificity.

VEH- and EDC-MMSCs were seeded onto coated-glass coverslips and culture under the conditions stated above. Confluent cells were then fixed with 4% paraformaldehyde for 15 min, followed by permeabilization with 0.1% Triton X-100 in PBS for 15 min at room temperature. Non-specific binding was blocked with 2% BSA in PBS for 1 h at room temperature. Primary antibody targeting TGFβ1 (MA5-15065, Invitrogen, 1:100 in 0.1% BSA-PBS) and TGFβ Receptor I (PA-95863, Invitrogen, 1:100 in 0.1% BSA-PBS) were applied and incubated overnight at 4 °C. The cells were then washed with PBS three times for 5 min and incubated with Alexa Fluor™ 568-conjugated α-Rabbit secondary antibody (A11011, Invitrogen, 1:2000 in 0.1% BSA-PBS) for 1 h at room temperature. Finally, the coverslips were mounted onto glass slides using mounting medium containing DAPI for nuclear counterstaining (H-1200, VECTASHIELD). Digital image files were created with an Olympus VS200 Research Slide Scanner (Olympus/Evident, Center Valley, PA) with a Hamamatsu ORca-Fusion camera (Hamamatsu Photonics, Skokie, IL). Individual images were created with the OlyVIA Viewer software (Olympus/Evident, Center Valley, PA). Negative controls without primary antibody were included to validate the staining specificity.