Cryosectioning and Immunostaining of Enucleated Eyeballs

Each enucleated eye ball was fixed with paraformaldehyde and then placed in a slurry of optimal cutting temperature (OCT) compound in cryomold before freezing in dry ice and storage in a − 80 °C freezer until ready for sectioning using the Microm HM550 (Carl Zeiss Ltd). Five-micrometer cryosections of day 2 post-operated eye tissues stained with hematoxylin and eosin (H&E) staining was visualized as described previously [14 (link)]. A total of 3 eyes for each condition were evaluated. Acetyl-histone H3, CD45 and F4/80 antibodies were obtained from Merck Millipore (Darmstadt, Germany), BD Pharmingen (San Diego, CA, USA) and Abcam Plc (Cambridge, UK), respectively. Isolectin B4-Alexa Fluor 568 conjugate was from Molecular Probes Inc. (Eugene, OR, USA). Labeling by the primary antibodies was detected using secondary antibodies conjugated to Alexa Fluor-594 (red fluorescence) or Alexa Fluor-488 (green fluorescence), both obtained from Invitrogen Corp. (Thermo Fisher Scientific Inc., Waltham, MA, USA). Nuclei were visualized by mounting the cells in DAPI-containing Vectashield mounting medium (Vector Laboratories, CA, USA). Labeled cells were visualized using the Zeiss Imager.Z1 microscope (Carl Zeiss Inc., USA).