The next day, membranes were washed in PBS T and incubated for 1 hour at room temperature with appropriate fluorescence conjugated secondary antibodies, donkey anti mouse IR Dye 680, goat anti rat IR Dye 680, and donkey anti rabbit IR Dye 800CW. Membranes were washed again in PBS T and the fluorescent bands were detected using LI CORs Odyssey selleck chem Perifosine infrared imaging system. Leukemia inhibitory factor ELISA A total of 1 mL of supernatant was collected from each well of the six well plates of primary mouse astrocyte cul tures, and these samples were stored at ?20 C. ELISA plates were coated overnight at room temperature with 100 ul well of primary antibody goat anti LIF diluted in 0. 01 M PBS. The following day, the plates Inhibitors,Modulators,Libraries were washed six Inhibitors,Modulators,Libraries times with wash buffer using an automated microplate washer and air dried.
Plates were subse quently incubated for 1 hour at room temperature with 200 Inhibitors,Modulators,Libraries ul well of blocking buffer. After blocking, the plates were incubated with superna tants from astrocyte cultures for 2 hours at room temperature. Two dilutions of each sample, diluted in incubation buffer, were made in triplicates. The plates were then incubated for 1 hour at room temperature with 100 ul well of the detection antibody, biotinylated goat anti LIF diluted in incubation buffer, followed by an incubation for 30 minutes at room temperature with 100 ul well of Streptavidin horseradish peroxidase conjugate. The plates were then incubated for 15 to 20 minutes at room temperature with 100 ul well of TMB detection buffer. Upon stable color formation the reactions were stopped by adding 100 ul well of 1 M H2SO4.
Absorbance of the samples was measured using VersaMax, a spectrophotometric ELISA plate reader, and SoftMax Pro software at 450 nm, with a background correction Inhibitors,Modulators,Libraries at 575 nm. Recombinant mouse LIF was used to plot the standard curve. MTT assay Survival of cultured embryonic cortical neurons or cultured neonatal astrocytes Inhibitors,Modulators,Libraries against various experi mental treatments was measured by the colorimetric http://www.selleckchem.com/products/Gefitinib.html MTT 2,5 diphenyltetra zolium bromide assay, as described previously. MTT solution was added to cultured cells and incubated for 4 hours, after which, cells were lysed and MTT formazan solu bilized in dimethyl sulfoxide on an orbital shaker for 15 minutes. Optical density measure of each sample was determined using an automated ELISA reader the Varioskan Flash spectral scanning multimode reader at 570 nm, with a background correction at 630 nm. Immunocytochemistry and confocal microscopy Astrocytes cultured on glass cover slips were fixed for 15 minutes in 4% paraformaldehyde. After several washes in PBS, the cells were blocked for 45 minutes with 5% normal goat serum in PBS containing 0. 1% TritonX.