Alexa 488 labeled phalloidin

Goat antibody to Wdr-1 and rabbit antibody to focal adhesion kinase (FAK) were obtained from Santa Cruz Biotechnology, while rabbit anticofilin-1 antibody was from Cell Signaling Technology. Alexa 488-labeled sheep antirabbit antibody, Alexa 647-labeled donkey antigoat antibody and Alexa 488-labeled phalloidin were purchased from Invitrogen. Apyrase, polylysine and prostaglandin E₁ (PGE₁) were obtained from Sigma-Aldrich. Fibrinogen was isolated as described before (9 (link)). The hypomorphic allele of Wdr-1 mice has been described before (7 (link)). The mutant mouse (Wdr-1^rd/rd) has a T>A transversion in the second dinucleotide of the intron 9 splice donor and it produces a mutant transcript containing a 6-bp in-frame deletion that results in a incorrectly folded, nonfunctional protein (7 (link)). A small amount of normal splicing produces some wildtype protein, resulting in a hypomorphic allele (Figure 1, Panels A and B). All animals were treated in accordance with the protocol approved by the Animal Care and Use Committee (IACUC) of Baylor College of Medicine.

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.