At the end of the follow-up period, mice were sacrificed, pancreata were
harvested, fixed, and embedded in paraffin 6 h post-injection with BrdU (100
mg/kg/bw). Sections were stained using antibodies to BrdU (Dako, M0744, 1:100),
Ki67 (Dako, M7240, 1:100), phosphohistone H3 (Millipore, 06–570, 1:250),
or insulin (Abcam, ab7842, 1:500), glucagon (Sigma, G2654, 1:8000), somatostatin
(Abcam, ab30788, 1:1000), β2M (Abcam, ab87483, 1:80), serum anti-ZnT8
(c-term), 1:500, serum anti-phogrin (C-term), 1:250 (gift from H. Davidson, Univ
Colorado) and appropriate secondary antibodies and counterstained with DAPI
(Sigma, D9564, 1:6600). Cell death was detected by TUNEL assay (ApopTag S7100;
Chemicon). At least 1,000–2,000 β-cell nuclei were counted per
animal, and data were expressed as a percentage of BrdU+, Ki67+, pHH3+ or TUNEL+
β-cells. Insulitis was evaluated as reported previously38 (link). β-cell mass was
evaluated by point-counting morphometry on immunofluorescence-stained sections
of the pancreas5 (link).
harvested, fixed, and embedded in paraffin 6 h post-injection with BrdU (100
mg/kg/bw). Sections were stained using antibodies to BrdU (Dako, M0744, 1:100),
Ki67 (Dako, M7240, 1:100), phosphohistone H3 (Millipore, 06–570, 1:250),
or insulin (Abcam, ab7842, 1:500), glucagon (Sigma, G2654, 1:8000), somatostatin
(Abcam, ab30788, 1:1000), β2M (Abcam, ab87483, 1:80), serum anti-ZnT8
(c-term), 1:500, serum anti-phogrin (C-term), 1:250 (gift from H. Davidson, Univ
Colorado) and appropriate secondary antibodies and counterstained with DAPI
(Sigma, D9564, 1:6600). Cell death was detected by TUNEL assay (ApopTag S7100;
Chemicon). At least 1,000–2,000 β-cell nuclei were counted per
animal, and data were expressed as a percentage of BrdU+, Ki67+, pHH3+ or TUNEL+
β-cells. Insulitis was evaluated as reported previously38 (link). β-cell mass was
evaluated by point-counting morphometry on immunofluorescence-stained sections
of the pancreas5 (link).