Tcs sp5 2 microscope

Dissection and labelling of retinas was performed as previously described27 (link). Briefly, retinas were fixed for 2 h on ice in 4% paraformaldehyde, incubated in 1% BSA and 0.3% Triton X-100, washed two times in Pblec (1% Triton X-100, 1 mM CaCl2, 1 mM MgCl2, and 1 mM MnCl2 PBS (pH 6.8)), and incubated overnight with isolectin-B4 (1:50) and antibodies diluted in Pblec. Images were acquired and processed using a Leica TCS SP5 II microscope, LAS Montage Imaging software (Leica) and the IMARIS Digital Imaging software (Biplane). Biotinylated Griffonia simplicifolia lectin I (IB4), (B-1205) was purchased from Vector Laboratories.

Using previously established methods^{8 (link)}, brains were post-fixed in 4% paraformaldehyde in PBS at 4° C for ten ^99mTc-albumin half-lives (~60 h). Hemisections were equilibrated in 30% sucrose in PBS for 24 h at 4 °C and embedded in OCT (Tissue-Tek, Torrance, CA). Antigen retrieval was performed with 50 mM sodium citrate (pH 9.0) and then heating at 80 °C for 30 min. Immunostaining was performed as previously described^{14 (link)}. Floating tissue sections were cover slipped with a drop of Prolong Gold Antifade Reagent. The following antibodies were used: rabbit polyclonal anti-EAAT4 (Abcam, Cambridge, MA; 1:1,000), mouse monoclonal calbindin (Abcam; 1:1,000), rabbit polyclonal anti-GLUT1 (Millipore, Billerica, MA; 1:500), and goat polyclonal anti-ICAM-1 (Novus Biologics, Centennial, CO; 1:1,000). Corresponding secondary antibodies labeled with Alexa 488, and Cy3 were applied for 2 h (Jackson Immunoresearch, West Grove, PA; 1:1,000). Confocal microscopy was performed with a Leica TCS SP5 II microscope. Microscopic images were acquired with the Leica Application Suite and processed using image adjustments limited only to linear contrast and brightness adjustments applied identically to data from blast- and sham-treated animals in each experiment.