The sections had been treated for equal time in DAB reagent and photographed simultaneously. Phosphorylated GluR1, phosphorylated ERK 1 SOCS3, BAX, and glutamine synthase have been detected applying Rhodamine labeled secondary antibody. Nuclei have been stained with DAPI. Sections were washed, mounted and viewed below a fluorescence microscope. As a unfavorable control, sections had been treated inside the same manner, except that incubation with principal anti physique was omitted. Isolation of retinal ganglion cell layer by laser capture microscopy The LCM system that we utilized obviated the need to have for tissue dehydration before microdissection. This enabled us to isolate quiescent retinal ganglion cells straight from 8M frozen sections of mouse eyes, thereby growing the yield and high-quality of RNA.
Fro zen sections, mounted on unique membrane coated slides, which facilitated the capture of cells, were briefly stained with hematoxylin. As well as visualizing the retinal structures, this staining procedure also removed the OCT mounting selleck medium. Labeled sec tions have been tracked with intergral light microscope employing a 20? objective. Retinal ganglion cells to become isolated were outlined having a light pen or cursor on a monitor screen. Such an outline defined the location that will be cut and catapulted intact into a Capsure Macro LCM cap. In this manner, approxi mately 6000 cells from the ganglion cell layer have been isolated from every eye. Total RNA Isolation and cRNA Amplification Total cellular RNA from LCM captured cells was isolated and purified. Samples from the total starting RNA had been analyzed by capillary electrophoresis to assess the degree of purification.
Around 60 ng of total cellular RNA may very well be extracted from 6000 cells from the ganglion cell layer that were isolated by LCM. When this RNA was con trasted with commercially prepared total RNA from mouse liver pop over to this site working with picogram chips plus a Bioanalyzer, sharp bands corresponding to the 18 S and 28 S RNA were observed for all samples RNA high-quality was further assessed by calculating the RNA integrity number, that is according to a proprietary Agilent Technol ogies algorithm. Total RNA in the isolated cells was subjected to cRNA amplification. Briefly, 1. 5 rounds of cRNA amplification were accomplished utilizing a Ribo Amp OA RNA amplification protocol. 1st strand cDNA was generated by reverse transcription working with the total RNA. Just after the second strand cDNA was synthe sized, a T7 RNA polymerase driven cRNA synthesis was performed to acquire the initial round of cRNA amplifica tion. A second double strand cDNA synthesis was per formed followed by a second round of cRNA amplification. A BioArray HighYield RNA transcript labeling protocol was employed for the second round of amplification to biotinylate the cRNAs.