Picture frame 3

Live embryos or in situ hybridization stainings were imaged using an Olympus SZ61 dissecting microscope with a high-resolution digital camera (model S97809, Olympus America) and Picture Frame 3.0 software (Optronics).
Semi-thin sections and flat-mounted embryos from Alcian blue staining were imaged using a Zeiss Axioskop2 plus microscope with a Zeiss AxioCam MRc camera and Zeiss AxioVision Rel. 4.6 software.
Live embryos, whole-mount and sections immunohistochemistry were visualized using an inverted Leica Sp2 AOBS confocal microscope using either 40x or 63x oil-immersion objective. For wholemount, embryos were embedded in 1% low-melt agarose (BP165-25, Fisher) prior to imaging. Images were acquired using Leica software.

At the end of the in vitro permeation studies, the corneas that were exposed to medium (control), TA-SLN, TA-SLN-IG and TA-C formulations were used for histology studies. The isolated corneas were fixed with 4% paraformaldehyde for about 2 to 3 h. Later the corneas were rinsed with PBS, embedded in paraffin wax and sectioned at 5-micron thickness using a microtome (American Optical^® 820 Rotary Microtome, Labequip, Torrance, Ontario, Canada). The tissue sections were mounted on a slide and dried overnight in an oven. The slide was washed with xylene to remove the paraffin and the tissue was hydrated by washing with alcohol and water. The tissue was stained in nuclear dye Gill III hematoxylin (StatLab medical, Baltimore, MD, USA) by rinsing for 10 min and later counterstained with eosin. The sections were stained with hematoxylin and eosin, viewed on an Eclipse 800 photomicroscope (Nikon, Melville, NY, USA) and the images were captured using Picture Frame 3.0 software (Optronics), to record the morphological changes in the treated cornea, if any [28 ].