Hanks balanced salt solution buffer

Primary MEFs grown on poly-d-lysine-treated coverslips were incubated on ice for 30 min in Hanks balanced salt solution buffer (Sigma) containing 1 μg/ml Alexa Fluor 555-labeled subunit B of cholera toxin (CTxB; Invitrogen). After unbound CTxB was washed, growth medium was added and the cells were incubated at 37°C for the times indicated below. After the chase period, cells were fixed with 4% paraformaldehyde in PBS and permeabilized with 0.1% Triton X-100. Immunolabeling of the Golgi apparatus was performed with an anti-GM130 mouse monoclonal antibody (35/GM130; BD Biosciences) and an Alexa Fluor 488-labeled anti-mouse IgG (Invitrogen). Coverslips were mounted on slides with ProLong Gold antifade reagent with DAPI (4′,6-diamidino-2-phenylindole; Invitrogen). Cells were imaged by confocal microscopy with appropriate excitation/emission settings for detection of Alexa Fluor 555, Alexa Fluor 488, and DAPI. The proportion of CTxB localized in the Golgi apparatus was calculated by producing binary layers by thresholding the CTxB and Golgi apparatus channels. Threshold parameters were kept constant for all cells analyzed, and the proportion of total CTxB colocalizing with the Golgi apparatus was calculated.

The corneas of the rats were harvested and washed twice with Hanks balanced salt solution buffer (Sigma Chemicals). After careful removal of excessive corneal stroma, the tissue was cut into small pieces, placed onto culture plate and cultured with medium containing Dulbecco’s modified Eagle medium (DMEM), 5 ng/ml of epidermal growth factor (EGF), 1% glutamax, 1% non-essential amino acids, 1% antibiotic solution and 5% FBS. The cultured CECs were identified by CK-3 immunofluorescence and the percentage of CK3-positive cells was examined by flow cytometry. CK3 antibody was from Biobyt (orb5866).