The sections have been treated for equal time in DAB reagent and photographed simultaneously. Phosphorylated GluR1, phosphorylated ERK 1 SOCS3, BAX, and glutamine synthase had been detected utilizing Rhodamine labeled secondary antibody. Nuclei had been stained with DAPI. Sections had been washed, mounted and viewed under a fluorescence microscope. As a damaging handle, sections had been treated within the same manner, except that incubation with primary anti physique was omitted. Isolation of retinal ganglion cell layer by laser capture microscopy The LCM technique that we used obviated the need to have for tissue dehydration prior to microdissection. This enabled us to isolate quiescent retinal ganglion cells straight from 8M frozen sections of mouse eyes, thereby increasing the yield and top quality of RNA.
Fro zen sections, mounted on particular membrane coated slides, which facilitated the capture of cells, had been briefly stained with hematoxylin. Along with visualizing the retinal structures, this staining procedure also removed the OCT mounting selleck medium. Labeled sec tions had been tracked with intergral light microscope employing a 20? objective. Retinal ganglion cells to become isolated have been outlined using a light pen or cursor on a monitor screen. Such an outline defined the area 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 single eye. Total RNA Isolation and cRNA Amplification Total cellular RNA from LCM captured cells was isolated and purified. Samples of the total beginning RNA were analyzed by capillary electrophoresis to assess the degree of purification.
About 60 ng of total cellular RNA could be extracted from 6000 cells in the ganglion cell layer that have been isolated by LCM. When this RNA was con trasted with commercially prepared total RNA from mouse liver inhibitor Pazopanib making use of picogram chips and a Bioanalyzer, sharp bands corresponding for the 18 S and 28 S RNA had been observed for all samples RNA excellent was additional assessed by calculating the RNA integrity quantity, that is according to a proprietary Agilent Technol ogies algorithm. Total RNA from the isolated cells was subjected to cRNA amplification. Briefly, 1. 5 rounds of cRNA amplification had been accomplished employing a Ribo Amp OA RNA amplification protocol. 1st strand cDNA was generated by reverse transcription working with the total RNA. Soon after the second strand cDNA was synthe sized, a T7 RNA polymerase driven cRNA synthesis was performed to obtain the very first 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.