Pan keratin

Paraffin-embedment and hematoxylin and eosin (H&E) stains were performed at the Histology core facility at the Icahn School of Medicine at Mount Sinai. Histologic changes were evaluated and scored by a pathologist blinded to the treatment group. For immunofluorescence, de-waxed paraffin-embedded colonic tissue sections were stained as previously described [17 (link), 19 (link)]. Briefly, tissue sections were incubated with primary antibodies against MUC2 (SantaCruz), and pan-keratin (Abcam) followed by the appropriate secondary antibodies (Life Technologies), and counterstained using 4’, 6’-diamidino-2-phenylindole (DAPI). All slides were examined on a Nikon Eclipse Ni series microscope and images captured at 4x, 10x and 20x magnifications.

To characterize the pterygium fibroblasts isolated from primary culture, the expression of keratin, Aldh3, and Prdx2 was analyzed by immunofluorescence. HPFs were fixed using 4% paraformaldehyde and stained for Aldh3a1 (1 : 100), Prdx2 (1 : 500), and pan-keratin (1 : 10) (Abcam, Cambridge, MA). Images were taken with a structured illumination microscope (Zeiss ApoTome-DZNE W1).

For histological staining, spheroids were collected and washed in DPBS, and fixed in 3.7% formaldehyde DPBS overnight, and then transferred to 30% sucrose for embedding into OCT medium for cryosectioning. Slices (6 µm thickness) were obtained using a Hyrax C20 (Carl Zeiss, Oberkochen, Germany) set at −26 °C. The staining was done according to the following immunohistochemistry procedure: Sections were washed in PBS and blocked with 2.5% goat serum, 0.1% Triton-X, 0.05% Tween20 in PBS, and then the samples were incubated with the primary antibody (Fibronectin, 1:300, Abcam; CD31, 1:100, Abcam; CD105 1:100, Santa Cruz Biotechnology; Pan-keratin, 1:100, Abcam) overnight and washed afterwards before counterstaining with hematoxylin. Control sections were processed without the primary antibody. Cultures grown in 2D were directly stained without the need for sectioning.
For live-dead staining (LIVE/DEAD Viability/Cytotoxicity Kit, Invitrogen, Waltham, MA, USA), spheroids were collected and washed twice with DPBS. DPBS was aspirated and cells were stained with 2 µM calcein AM (live) and 5 µM ethidium homodimer-1 (EthD-1, dead) DPBS solution for 30–60 min. After the incubation, cells were washed with DPBS once and imaged.