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Schlagwörter:
laminin; immunofluorescence; RT-PCR; radioimmuno-inhibition assay; monoclonal antibody; epitope; masking
Zusammenfassung:
Protein levels, mRNA expression, and localization of laminin alpha1 and alpha2 chains in development and in adult mice were examined. Recombinant fragments were used to obtain high-titer- specific polyclonal antibodies for establishing quantitative radioimmuno-inhibition assays. This often demonstrated an abundance of alpha2 chain, but also distinct amounts of alpha1 chain for adult tissues. The highest amounts of alpha1 were found in placenta, kidney, testis, and liver and exceeded those of alpha2. All other tissue extracts showed a higher content of alpha2, which was particularly high in heart and muscle when compared to alpha1. Content of gamma1 chain, shared by most laminins, was also analyzed. This demonstrated gamma1 chain levels being equal to or moderately exceeding the sum of alpha1 and alpha2 chains, indicating that these isoforms represent the major known laminin isoforms in most adult mouse tissues so far examined. Moreover, we found good correlation between radioimmuno-inhibition data and mRNA levels of adult tissues as measured by quantitative real-time reverse transcriptase-PCR. Embryonic tissues were also analyzed by radioimmuno-inhibition assays. This demonstrated for day 11 embryos comparable amounts of alpha1 and gamma1 and a more than 25-fold lower content of alpha2. This content increased to about 10% of alpha1 in day 13 embryos. The day 18 embryo showed in heart, kidney, and liver, but not yet in brain and lung, alpha1/alpha2 chain ratios comparable to those in adult tissues. Immunostaining demonstrated alpha1 in Reichert's membrane (day 7.5), while alpha2 could not be detected before day 11.5. These data were compared with immunohistochemical localization results on several more embryonic and adult tissue sections. Our results regarding localization are consistent with those of earlier work with some notable exceptions. This was in part due to epitope masking for monoclonal antibodies commonly used in previous studies in esophagus, intestine, stomach, liver, kidney, and spleen. (C) 2002 Elsevier Science (USA).
