The rats were then euthanized, http://www.selleckchem.com/products/PD-0332991.html and the retinas Inhibitors,Modulators,Libraries were collected for analysis by immunoblotting or histology. Eyes were removed and opened by circumferential incision just below Inhibitors,Modulators,Libraries the ora serrata, and anterior segment and the vitreous were dis carded. Under a dissection microscope, the retina was gently lifted off the eyecup. H E staining Retinas were immersion fixed in 4% formaldehyde, dehydrated through graded ethanol steps and xylene, then embedded in paraffin. Sections were cut with a vibrotome at a thickness of 5 um and mounted onto glass slides. The mounted sections were deparaffinized with xylene and rehydrated with graded ethanol steps from 100% to 70%. Hematoxylin was used to stain the sections for 3 min, followed by washing with tap water. After treatment with 0. 1% HCl and 0.

1% NH4OH, sections were exposed to eosin for 3 min, then dehydrated with graded ethanol steps and xylene, and coverslipped in neutral balsam. Observations were made under phase contrast and bright field microscopy. TUNEL staining Apoptosis was analyzed with the In Situ Cell Death Detection Kit. Frozen sections of the rat retinas were cut on a cryostat. The sections were postfixed with Inhibitors,Modulators,Libraries 4% paraformaldehyde and permeablized with 0. 1% Tri ton X 100. A 50 ul TUNEL reaction mixture was added to each sample, and the slides were incubated in a humidified atmosphere for 60 min at 37 C in the dark and analyzed by fluorescence microscopy with an FITC filter. Western blotting for rat retinal homogenates Retinas were homogenized with protein lysis buffer con taining protease inhibitor cocktail and then centrifuged at 13,000 �� g at 4 C for 10 min to remove insoluable pellets.

The supernatants were quantified with BCA reagents. Retinal proteins from control or STZ Inhibitors,Modulators,Libraries injected rats were loaded in individual lanes, resolved with SDS PAGE analysis, and then electrophoretically transferred to a nitrocellulose membrane. The transfer efficiency was monitored with Ponceau S, and blots were blocked with 3% BSA or skim milk. SR antibody or JNK phospho JNK antibody was diluted in Tris buffered saline with 0. 1% Tween 20 sup plement and applied to the blots overnight at 4 C. Following washes with TBS, a peroxidase conju gated secondary antibody was applied at a dilution of 1,5000. Washes were followed by development with Pierce ECL Western Blotting Substrate.

Each membrane probed for SR or JNK was stripped and probed for GAPDH detection. Immunofluorescence Frozen sections of retina were blocked with skimmed milk overnight. SR antibody in PBS containing 0. 1% Triton X 100 was applied to the sections for 1 h at room temperature then overnight at 4 C. On the following day, Inhibitors,Modulators,Libraries the samples were washed three times with novel PBS and incubated for 1 h at room temperature with a secondary antibody conjugated to Alex Fluor 488. Following incubation in secondary antibody, the sections were washed in PBS at 4 C, coverslipped, and examined with a Zeiss Axiovert 200 equipped with epifluorescence optics.