Objective To detemine optimal conditions by using 5 bromodeoxyuridine (BrdU) as a marker of transplanted retinal pigment epithelial (RPE) cells in the subretinal space of albino rabbits ...Objective To detemine optimal conditions by using 5 bromodeoxyuridine (BrdU) as a marker of transplanted retinal pigment epithelial (RPE) cells in the subretinal space of albino rabbits Methods Pigmented rabbit RPE cells at second to fifth passage were fed with 20 μmol/L BrdU in Eagle’s minimal essential medium (MEM) for five days After extensive wash with phosphate buffered saline (PBS),the cells were detached by trypsin and used for transplantation onto Bruch’s membrane of albino rabbits Eyes were enucleated at various times post transplantation Acetone, 4% paraformaldehyde, periodate lysine paraformaldehyde (PLP),or half strength Karnovsky’s fixatives were individually used to fix the tissue in order to find optimal condition for detecting BrdU marker The fixation was followed by embedding in OCT compound, glycol plastic, or paraffin The transplanted area was then sectioned, pepsin digested, and used for immunohistochemical staining with monoclonal antibody against BrdU and avidin biotin alkaline phosphatase complex (ABC AP) Results Frozen sections of acetone or paraformaldehyde fixed tissue gave strong immunostaining of BrdU but the overall morphology was poor Karnovsky’s fixed tissue offered strong staining but this was buried by strong background When using PLP as a fixative, we obtained strongly positive blue staining with very low background, and also excellent morphologic preservation Conclusion In combination with immunohistochemical detection method, BrdU labeling is an excellent long term marker for RPE transplantation one year after surgery To use BrdU as a marker necessitates the use of pepsin digestion to make the BrdU antigen in the nuclei accessible to antibody But the pepsin digestion may damage other tissues and result in overall poor morphology Among the fixatives tested in this study, PLP fixed tissues offered both strong BrdU staining and good preservation of structural integrity, particularly the fine structure of photoreceptor and RPE cells展开更多
文摘Objective To detemine optimal conditions by using 5 bromodeoxyuridine (BrdU) as a marker of transplanted retinal pigment epithelial (RPE) cells in the subretinal space of albino rabbits Methods Pigmented rabbit RPE cells at second to fifth passage were fed with 20 μmol/L BrdU in Eagle’s minimal essential medium (MEM) for five days After extensive wash with phosphate buffered saline (PBS),the cells were detached by trypsin and used for transplantation onto Bruch’s membrane of albino rabbits Eyes were enucleated at various times post transplantation Acetone, 4% paraformaldehyde, periodate lysine paraformaldehyde (PLP),or half strength Karnovsky’s fixatives were individually used to fix the tissue in order to find optimal condition for detecting BrdU marker The fixation was followed by embedding in OCT compound, glycol plastic, or paraffin The transplanted area was then sectioned, pepsin digested, and used for immunohistochemical staining with monoclonal antibody against BrdU and avidin biotin alkaline phosphatase complex (ABC AP) Results Frozen sections of acetone or paraformaldehyde fixed tissue gave strong immunostaining of BrdU but the overall morphology was poor Karnovsky’s fixed tissue offered strong staining but this was buried by strong background When using PLP as a fixative, we obtained strongly positive blue staining with very low background, and also excellent morphologic preservation Conclusion In combination with immunohistochemical detection method, BrdU labeling is an excellent long term marker for RPE transplantation one year after surgery To use BrdU as a marker necessitates the use of pepsin digestion to make the BrdU antigen in the nuclei accessible to antibody But the pepsin digestion may damage other tissues and result in overall poor morphology Among the fixatives tested in this study, PLP fixed tissues offered both strong BrdU staining and good preservation of structural integrity, particularly the fine structure of photoreceptor and RPE cells
