horseradish peroxidase conjugated anti rabbit IgG secondary antibody was from Amersham Biosciences. All other chemicals had been of analytical grade. Cell culture The PAN02 murine pancreatic adenocarcinoma cell line was obtained from the National Cancer Institute and maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum. two mM L glutamine, one hundred U ml penicillin, and 100 ug ml streptomycin. Major cultured mouse skin fibroblasts from wild style and AT2 KO mice have been prepared from 24 to 48 hour previous C57BL 6J mouse pups following an estab lished process. MSFs had been cultured in DMEM Hams F twelve medium supplemented with 10% FBS, one hundred U ml penicillin, and a 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 were back crossed with wild sort C57BL 6J for 17 generations such the genetic background in the mice is vulnerable to our pancreatic cancer syngeneic model. Wild sort littermates served as controls. Genotypes had been confirmed through the PCR system implementing extracted tail DNA. Briefly, published sequences were utilised to synthesize primers GSK2118436 distributor to the AT2 receptor plus the neomycin resistance gene products. Extracted tail DNA was amplified at 95 C for 1 minute. at 58 C for 1 minute. and at 72 C for one minute with 0. five nmol L of each primer, one. 25 units DNA polymerase, and 0. two mmol L deoxynucleotide triphosphates in PCR buffer. PCR pro ducts within the AT2 receptor and Neo r gene pro duct had 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 sort, heterozygote, and AT2 KO, respectively. All animals had been maintained in a humidity and temperature controlled space on twelve hour light dark cycles.
All procedures for handling animals were selleck inhibitor accepted from the Institutional Com mittee for Animal Care and Use of Kansas State University. Pancreatic cancer syngeneic model Seven to 9 week old AT2 KO C57BL 6J mice and wild style littermates have been anesthetized with isoflurane. Cells had been trypsinized and washed with PBS. Five mil lion cells in 200 ul PBS have been subcutaneously inoculated into each and every flank making use of a one ml syringe that has a 27G needle. The tumor size was measured by caliper each and every three days and also the volume was calculated applying the for mula two ? ? 0. 5. On the end with the experiments, the mice were sacri ficed by cervical dislocation below anesthesia. The tumors have been dissected and weighed. For histological assessment, the specimens had been fixed in 10% formalin, embedded in paraffin, and sectioned for histopathologi cal analysis. Immunohistochemical examination Tissue sections of four um thickness were ready for all staining. Slides have been dewaxed and rehydrated before staining.