horseradish peroxidase conjugated anti rabbit IgG secondary antibody was from Amersham Biosciences. All other chemical substances had been of analytical grade. Cell culture The PAN02 murine pancreatic adenocarcinoma cell line was obtained from the Nationwide Cancer Institute and maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum. two mM L glutamine, a hundred U ml penicillin, and 100 ug ml streptomycin. Primary cultured mouse skin fibroblasts from wild sort and AT2 KO mice have been prepared from 24 to 48 hour old C57BL 6J mouse pups following an estab lished technique. MSFs had been cultured in DMEM Hams F twelve medium supplemented with 10% FBS, a hundred U ml penicillin, and one hundred ug ml streptomycin. All cells were incubated in 5% CO2 humidified air at 37 C. Animals and genotyping Hemizygous AT2 KO mutant mice had been gener ated as described previously.
These mice have been back crossed with wild form C57BL 6J for 17 generations such that the genetic background within the mice is susceptible to our pancreatic cancer syngeneic model. Wild sort littermates served as controls. Genotypes had been confirmed from the PCR procedure utilizing extracted tail DNA. Briefly, published sequences were utilized to synthesize primers find more information for the AT2 receptor plus the neomycin resistance gene product or service. Extracted tail DNA was amplified at 95 C for one minute. at 58 C for one minute. and at 72 C for one minute with 0. five nmol L of each primer, 1. 25 units DNA polymerase, and 0. 2 mmol L deoxynucleotide triphosphates in PCR buffer. PCR pro ducts on the AT2 receptor and Neo r gene professional duct have been visualized by 1% agarose gel electrophoresis. AT2 and Neo r. AT2 and Neo r. and AT2 and Neo r had been assigned as wild type, heterozygote, and AT2 KO, respectively. All animals were maintained inside a humidity and temperature controlled room on 12 hour light dark cycles.
All procedures for handling animals have been pop over to this website approved through the Institutional Com mittee for Animal Care and Utilization of Kansas State University. Pancreatic cancer syngeneic model Seven to nine week previous AT2 KO C57BL 6J mice and wild kind littermates had been anesthetized with isoflurane. Cells were trypsinized and washed with PBS. 5 mil lion cells in 200 ul PBS have been subcutaneously inoculated into each and every flank implementing a 1 ml syringe with a 27G needle. The tumor size was measured by caliper every 3 days and also the volume was calculated employing the for mula two ? ? 0. 5. On the end in the experiments, the mice had been sacri ficed by cervical dislocation below anesthesia. The tumors have been dissected and weighed. For histological assessment, the specimens were fixed in 10% formalin, embedded in paraffin, and sectioned for histopathologi cal evaluation. Immunohistochemical evaluation Tissue sections of 4 um thickness had been ready for all staining. Slides have been dewaxed and rehydrated ahead of staining.