Immunofluorescence Analysis of hCECs

The hCECs were seeded in 384-well plates pre-coated with iMatrix-511 at a ratio of 500 cells/mm² and cultured in BGM alone (control) or BGM containing TGF-β1, 2, or 3, or one of the TGF-β cell signaling pathway inhibitors, for four weeks. The IF protocol was previously described by our team [29 (link),42 (link)]. Briefly, cells were fixed in pure methanol at room temperature for 15 min after rinsing with PBS containing Ca²⁺ and Mg²⁺. The cells were then rehydrated in PBS and incubated in blocking buffer (PBS, 2% bovine serum albumin, 2% goat serum) for 30 min at 37 °C. The primary antibodies, diluted to 1/300 in blocking buffer, were incubated with cells at 37 °C for one hour under gentle agitation (30 rpm). After three rinses in PBS, the secondary antibodies, diluted to 1/600, and DAPI diluted at 2 µg/mL in blocking buffer were incubated with cells at 37 °C for one hour under gentle agitation. After three rinses in PBS, the cells were immersed in Fluoromount-G^TM mounting medium (00-4958-02, Invitrogen) to protect the fluorochromes (Alexa Fluor™ 488 and DAPI). An epifluorescence inverted microscope (IX81, Olympus, Tokyo, Japan) with the CellSens software (Soft Imaging System GmbH, Olympus) was used to acquire images.