Immunofluorescence Characterization of Proinsulin Processing in Human Fetal and Adult Pancreas

Abstract

Immunofluorescent staining is one of the most widely used techniques in basic research, yet the specificity and cross-reactivity of commercially available antibodies is often not well characterized. For instance, proinsulin processing has been extensively studied in the diabetes field by immunostaining, yet a collection of antibodies that are validated to be specific for each peptide(i.e. proinsulin, proinsulin intermediates, mature insulin and mature C-peptide) has not yet been described. This study aimed first to fully characterize the specificity of proinsulin processing antibodies and second, to study proinsulin processing during human fetal pancreas development. Immunofluorescent staining was performed on paraffin-embedded pancreas sections from several species, various transgenic mouse lines(lacking a hormone involved in proinsulin processing) and human fetal pancreas (9-21 weeks of gestation). Findings for antibody specificity are summarized in the table below. Briefly, the MAb1 antibody was specific for mature insulin, GS-9A8 for proinsulin or des-64,65 proinsulin intermediate, GN-ID4 for human proinsulin/C-peptide, Ab657 for mouse C-peptide 1 and Ab660 for mouse C-peptide2.Using these antibodies, we observed the presence of mature insulin in human fetal pancreas as early as 9 weeks of gestation, although there appeared to be differential proinsulin processing within the human fetal beta cells. In summary, we have developed an immunostaining-based tool to study proinsulin processing and demonstrated its utility by characterizing proinsulin processing in human fetal pancreas. Table Antibody Human Pig Dog Mouse PC1 ko PC2 ko CPE ko Furin ko Ins1 ko Ins2 ko Guinea pig anti-Insulin (Sigma) +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ Mouse anti-Insulin (MAb1) +++ - +/- +++ +++ ++ +/- + ++ +++ Mouse anti-Proinsulin (GS-9A8) +++ +/- + ++ +++ + ++ ++ ++ ++ Mouse anti-Proinsulin (GN-ID4) +++ + ++ - N/A N/A N/A N/A N/A N/A Rabbit anti-C-Peptide1 Ab657 - - - +++ +++ ++ ++ ++ - +++ Rabbit anti-C-Peptide2 Ab660 + - - +++ +++ ++ ++ ++ +++ - Open table in a new tab