Tissue teck

Briefly, corneal tissue blocks were embedded in O.C.T. Compound (Tissue-Teck, Sakura Finetek, Torrance, CA), snap frozen in liquid nitrogen, and stored in an ultralow freezer (−80 °C, Revco Ultima II, Asheville, NC) before sectioning with a Leica CM 1850 cryotome (Leica, Wetzlar, Germany). Nine 8-μm-thick tissue sections were collected at 100-μm intervals and placed onto glass slides, alternating between slides so that the greatest depth of sampling was achieved (three sections/slide). Sections on one slide were then stained with Alexa 488-labeled phalloidin (Invitrogen, Eugene, OR) for 20 min. Slides were washed three times for 5 min each, counterstained with DAPI (300 nM, Invitrogen) in PBS for 15 min, and then washed for a final 5 min. Coverslips were then added to the slides using Gel/Mount (Biomeda, Foster City, CA) mounting media.

After overnight fixing, a 3 mm wide, central strip of cornea was
isolated by trimming and placed in cold (4 °C) sucrose (15% w/v)
solution for 4 h. The corneal strip was then transferred to 30% (w/v)
sucrose and stored overnight at 4 °C. The corneal tissue was
subsequently embedded in O.C.T. Compound (Tissue-Teck, Sakura Finetek,
Torrance, CA), snap-frozen in liquid nitrogen and stored at −80
°C (Revco Ultima II ultrafreezer, Asheville, NC). The tissue was then
sectioned (Leica CM 1850 crytome, Leica, Wetzlar, Germany) to produce six
8-μm-thick tissue sections collected at 100 μm intervals.
Three sections were mounted onto a glass slide, and stained for 1 h with
Alexa 488 Phalloidin (Invitrogen, Eugene, OR) using 10 units per section
diluted in PBS. Sections were then counterstained with
4’,6-diamidino-2-phenylindole (DAPI) (300 nM,
Sigma-Aldrich) in PBS for 15 min and a coverslip was subsequently placed
over the tissue sections using Gel/Mount (Biomeda, Foster City, CA) mounting
media.