Lsm 5 exciter laser scanning confocal system

The Thy1-CFP mice were anesthetized with isoflurane (2%; MWI Veterinary Supply, Inc., Meridian, ID, USA) in 95% O₂ and 5% CO₂, and placed on a temperature-controlled heated platform at 38°C. Topical tropicamide and 1% atropine sulfate ophthalmic solution (both Bausch & Lomb Pharmaceuticals) were used to dilate the pupils. A drop of hypromellose (2.5%; Akorn Pharmaceuticals, Lake Forest, IL, USA) was placed in the eye and a glass coverslip was placed over it to create a planoconcave lens on the mouse cornea for in vivo imaging. An LSM 5 Exciter Laser scanning confocal system (Carl Zeiss Inc., Thornwood, NY, USA) was modified (using an Axioskop 2 upright microscope equipped with a LSM 5 Exciter Laser scanning confocal system) to visualize CFP [blue-light cSLO (bCSLO); 460 nm excitation and 490 nm detection]. The number of CFP+ RGCs in the same area of the retina were assessed in vivo once a week for 6 weeks.