Journal: Diabetologia

Article Title: Quantitative analysis of islet prohormone convertase 1/3 expression in human pancreas donors with diabetes

doi: 10.1007/s00125-024-06275-5

Figure Lengend Snippet: PC1/3 expression in alpha cells. ( a ) Representative images showing PC1/3, glucagon and somatostatin expression in islets from donors with or without diabetes. nPOD case numbers are indicated in the merged images. Scale bar, 100 μm. ( b ) Proportional glucagon-positive area in islets. ( c ) Median RFU of glucagon in islets. ( d – f ) Percentage of glucagon-positive, PC1/3-positive ( d ), glucagon-positive, PC1/3-negative ( e ) and glucagon-positive islet cells ( f ) in islets. In ( f ) the darker columns represent PC1/3-negative cells, the lighter columns represent PC1/3-positive cells. ( g ) Mean RFU of glucagon in glucagon-positive alpha cells. ( h ) Mean RFU of glucagon in PC1/3-positive vs PC1/3-negative cells. ( i ) Median RFU of PC1/3 in PC1/3-positive, glucagon-positive alpha cells. ( j ) Mean PC1/3 and glucagon co-localisation in islets. Data are shown for islets from non-diabetic donors (filled circles, older adults; empty circles, young adults), AAb + donors (filled circles, double AAb + ; empty circles, single AAb + ), donors with recent-onset type 1 diabetes (duration <5 years), donors with long-duration type 1 diabetes (>15 years) and donors with type 2 diabetes. Each data point represents the average value from one donor. Hatched bars show data for images analysed at the islet level; non-hatched bars show data for images analysed at the single-cell level. Normally distributed data ( b , g , h , j ) are expressed as mean ± SEM, non-normal data ( c , d , e , f , i ) are expressed as median ± IQR. * p <0.05, ** p <0.01 and *** p <0.001. GCG, glucagon; ND, non-diabetic; T1D, type 1 diabetes; T2D, type 2 diabetes; y, years

Article Snippet: A previously reported protocol [ ] was used, with the following primary antibody cocktails reconstituted in an antibody diluent (S080983-2; Agilent Technologies, CA, USA): (1) insulin (IR002, Agilent Technologies, RRID:AB_2800361, pan-insulin antibody, 1:4 dilution), proinsulin (GS - 9A8 , Developmental Studies Hybridoma Bank, IA, USA, RRID:AB_532383, detects the B-C junction of proinsulin, 1:50 dilution), PC1/3 (ab191452, Abcam, Cambridge, UK, RRID:AB_3064864, 1:500 dilution, likely detects both 87 kDa and 66 kDa forms of PC1/3); and (2) glucagon (ab92517, Abcam, RRID:AB_10561971, 1:1000 dilution), somatostatin (NB100–64650, Novus Biologicals, CO, USA, RRID:AB_965456, 1:200 dilution) and PC1/3 (ab191452, Abcam, RRID:AB_3064864, 1:500 dilution).

Techniques: Expressing

Journal: Diabetologia

Article Title: Quantitative analysis of islet prohormone convertase 1/3 expression in human pancreas donors with diabetes

doi: 10.1007/s00125-024-06275-5

Figure Lengend Snippet: Glucagon and somatostatin expression pattern in islets. ( a ) Representative images showing PC1/3, glucagon and somatostatin expressions in islets from a donor without diabetes. Yellow arrows indicate the cytoplasmic processes of delta cells. Scale bar, 100 μm. ( b – d ) Proportional somatostatin-positive area ( b ), median RFU of somatostatin ( c ) and co-localisation of PC1/3 and somatostatin ( d ) in islets from non-diabetic donors (filled circles, older adults; empty circles, young adults), AAb + donors (filled circled, double AAb + ; empty circles, single AAb + ), donors with recent-onset type 1 diabetes (duration <5 years), donors with long-duration type 1 diabetes (>15 years) and donors with type 2 diabetes. Each data point represents the average value from one donor. Data are expressed as median ± IQR. * p <0.05 and ** p <0.01. GCG, glucagon; ND, non-diabetic; SST, somatostatin; T1D, type 1 diabetes; T2D, type 2 diabetes; y, years

Article Snippet: A previously reported protocol [ ] was used, with the following primary antibody cocktails reconstituted in an antibody diluent (S080983-2; Agilent Technologies, CA, USA): (1) insulin (IR002, Agilent Technologies, RRID:AB_2800361, pan-insulin antibody, 1:4 dilution), proinsulin (GS - 9A8 , Developmental Studies Hybridoma Bank, IA, USA, RRID:AB_532383, detects the B-C junction of proinsulin, 1:50 dilution), PC1/3 (ab191452, Abcam, Cambridge, UK, RRID:AB_3064864, 1:500 dilution, likely detects both 87 kDa and 66 kDa forms of PC1/3); and (2) glucagon (ab92517, Abcam, RRID:AB_10561971, 1:1000 dilution), somatostatin (NB100–64650, Novus Biologicals, CO, USA, RRID:AB_965456, 1:200 dilution) and PC1/3 (ab191452, Abcam, RRID:AB_3064864, 1:500 dilution).

Techniques: Expressing

Journal: Cell transplantation

Article Title: Intracameral Microimaging of Maturation of Human iPSC Derivatives into Islet Endocrine Cells.

doi: 10.1177/09636897211066508

Figure Lengend Snippet: Figure 4. In vitro confocal microscopic characterization of islet hormone immunofluorescence in single hiPSC-SIs before and after intracameral transplantation. A, Representative insulin immunofluorescence (red) and DAPI fluorescence (blue) in the cryosection of an intact single intracameral hiPSC-SI on the iris of an eyeball retrieved at 90 dpt. B, Sample insulin (red), glucagon (white), somatostatin (green) immunofluorescence and DAPI fluorescence (blue) in a cryosectioned hiPSC-SI before intracameral transplantation. C, An example image of insulin (red), glucagon (white), somatostatin (green) immunofluorescence and DAPI fluorescence (blue) in a cryosection of an intact single intracameral hiPSC-SIs retrieved at 90 dpt. D, Quantitative analysis of insulin immunofluorescence in hiPSC-SIs not subjected to intracameral transplantation as control (n ¼ 8) and intraoclular hiPSC-SIs retrieved from eyeballs at 90 dpt (n ¼ 8). Herein, n denotes the number of hiPSC-SIs, one of which comes from one recipient mouse. Each of four different batches of hiPSC-SIs was transplanted into two recipients. *P < 0.05 vs. control. a.u., arbitrary units.

Article Snippet: Subsequently, some specimens were triple-labeled with rabbit polyclonal antibody to glucagon (1:500; BioGenex, Fremont, CA, USA), guinea pig polyclonal antibody to insulin (1:1000, Dako North America, Carpinteria, CA, USA) and rat monoclonal antibody to somatostatin (1:1000; Novus Biologicals, Centennial, CO, USA).

Techniques: In Vitro, Immunofluorescence, Transplantation Assay, Fluorescence, Control