Alexa fluor 488 or alexa 555 conjugated secondary antibodies

Enucleated eyes were fixed overnight in 4% PFA in PBS at 4 °C, followed by dehydration and paraffin embedding. De-paraffinized and rehydrated tissue sections (5 μm) were stained with Hematoxylin and Eosin and examined under a stereomicroscope (EVOSFL Auto, life technologies). For immunofluorescent staining, tissue sections were de-paraffinized, rehydrated and subjected to antigen retrieval in sodium citrate buffer (10 mM sodium citrate, 0.05% Tween-20, pH 6.0) at boiling temperature for 30 minutes. Corneal sections were then blocked with 3% bovine serum albumin (BSA) in PBS containing 0.05% NP-40 for 1 hour at room temperature, then incubated overnight at 4 °C with the primary antibodies diluted in the same buffer. After three washes in PBST (PBS/0.1% Tween-20), slides were incubated at room temperature for 1 hour with Alexa Fluor 488- or Alexa 555-conjugated secondary antibodies (Invitrogen) and 1 μg/ml DAPI (Cat: #D3571; Molecular Probes, Inc. Eugene, OR) as a nuclear counterstain, washed with PBST again, and mounted with Mowiol (Sanofi-Aventis U.S.). Sections were photographed using a Zeiss microscope equipped with a camera (Axiocam Mrm). For data acquisition, we used the Axiovision 4.6 software (Carl Zeiss). The specifications for the antibodies used in this study are listed in supplementary Table 2.

Enucleated eyes and eyelids were fixed overnight in 4% PFA in PBS at 4 °C, followed by dehydration and paraffin embedding. De-paraffinized and rehydrated tissue sections (5 μm) were stained with Hematoxylin and Eosin and examined under a stereomicroscope (EVOSFL Auto, Life Technologies, Carlsbad, CA, USA). For immunofluorescent staining, tissue sections were subjected to antigen retrieval in sodium citrate buffer (10 mM sodium citrate, 0.05% Tween-20, pH 6.0) at boiling temperature. Cornea sections were then blocked with 3% bovine serum albumin (BSA) in PBS containing 0.05% NP-40 for 1 h at room temperature, then incubated overnight at 4 °C with the primary antibodies diluted in the same buffer. After three washes in PBST (PBS/0.1% Tween-20), slides were incubated at room temperature for 1 h with Alexa Fluor 488- or Alexa 555-conjugated secondary antibodies (Invitrogen, Waltham, MA, USA) and 1 μg/mL DAPI (Cat: #D3571; Molecular Probes, Inc. Eugene, OR, USA) as a nuclear counterstain, washed with PBST again, and mounted with Mowiol (Sanofi-Aventis U.S., Bridgewater, NJ, USA).