Introduction
Pancreatic islet cells are highly differentiated and can be characterized by the distinctive hormones which they produce: insulin for beta cells, glucagon for alpha cells, somatostatin for delta cells and pancreatic polypeptide for PP cells (Baskin, 2015). Five recombinant antibodies (AK247, AK249, AK250, AK280 and AK281) detect pancreatic glucagon-secreting alpha-cells by immunohistochemistry and one (AK251) does not.
Materials & Methods
Antibodies: ABCD_AK247, ABCD_AK249, ABCD_AK250, ABCD_AK251, ABCD_AK280 and ABCD_AK281 antibodies (ABCD nomenclature, http://web.expasy.org/abcd/; Lima et al., 2020) were produced by the Geneva Antibody Facility (http://unige.ch/medecine/antibodies/) as mini-antibodies with the antigen-binding scFv fused to a rabbit IgG Fc. The synthesized scFv sequences (GeneArt, Invitrogen) correspond to the sequences of the variable regions joined by a peptide linker (GGGGS)3 (see Table 1 for clone names and references). HEK293 suspension cells (growing in FreeStyleTM 293 Expression Medium; Gibco, #12338) were transiently transfected with the vector coding for the scFv-Fc of each antibody. Supernatants (see Table 1 for individual yields) were collected after 4 days.
| ABCD | Clone | Reference | Yield ( mg /L) |
| AK247 | H4H10223P | Okamoto and Gromada, 2016 | 5 |
| AK249 | H4H10232P | Okamoto and Gromada, 2016 | <5 |
| AK250 | H4H10236P | Okamoto and Gromada, 2016 | 100 |
| AK251 | H4H10237P | Okamoto and Gromada, 2016 | 40 |
| AK280 | IBA009 | Watson et al., 2014 | 70 |
| AK281 | IBA032 | Watson et al., 2014 | 100 |
Antigen: All antibodies were originally raised in mice against human glucagon (Uniprot #P01275) (Okamoto and Gromada, 2016; Watson et al., 2014).
Protocol: Mouse pancreas was surgically removed, fixed in PBS + 4% paraformaldehyde during 2 hs at RT. Pancreas sections were performed at the Histology Core Facility of the Geneva medical school, Switzerland. First, pancreas samples were dehydrated in an automatic tissue processing machine (Histokinette, Leica) in successive baths of ethanol at growing concentrations (70% twice 2 hs, 90% once 1 h, 95% once 1 h, 100% three times 1 h), followed by incubation in a solvent bath (three times 1 h; HistoSav, Biosystems #39-0591-05) and in bathes of liquid paraffin (three times 1 h; Leica #39601006). Samples were then embedded in liquid paraffin, cooled until solidification, and sectioned into 5 µm thick slices. Sections were deparaffinized and rehydrated with a series of alcohol solutions of decreasing concentrations (5 min in each solution, 100%, 95%, 70% and 50%) and kept in phosphate buffer saline (PBS) 30 min. Antigen retrieval was performed on deparaffined sections immersed in citrate 10 mM in PBS, pH 6 and microwave heated (once 7 min at 650 W and twice 5 min at 350 W). After blocking the endogenous peroxidase for 10 min with hydrogen peroxide (35%) diluted in 100% methanol, slides were washed for 3 min in ethanol (100%) and for 10 min in PBS. Sections were incubated for 30 min at RT with the primary antibodies (1/10) in antibody dilution buffer (DAKO #S2022). After three washes (5 min) in PBS, slides were incubated 20 min at RT with biotin-coupled anti -rabbit IgG (DAKO #K5001). Slides were then washed two times for 5 min in PBS and incubated for 20 min at RT with horseradish peroxidase-coupled streptavidin. After two washes (5 min) in PBS, slides were incubated with DAB chromogenic substrate (DAKO #K5001) until signal formation. Slides were then washed two times for 5 min in PBS and counter-stained with hemalum for 2 min. Sections were mounted in aqueous mounting medium (Aquatex, Sigma- Aldricht #108562) and scanned with an Olympus VS120 microscope.
Results
Alpha cells of pancreatic islets were successfully stained by AK247, AK249, AK250, AK280 and AK281 antibodies directed against glucagon, showing a strong intracellular signal (Fig. 1). No staining was observed with the AK251 antibody. The staining observed with the AK247 antibody confirms previously published observations (Frances and Thorel, 2019). No staining was observed in pancreas sections in which the primary antibody was omitted (Fig. 1, negative control).
Figure 1. AK247, AK249, AK250, AK280 and AK281 antibodies bind specifically to pancreatic glucagon-secreting alpha cells, as detected by immunohistochemistry. No staining was observed with the AK251 antibody. No staining was observed when the primary antibody was omitted (negative control). Scale bar: 50 µm.
Conflict of interest
The authors declare no conflict of interest.