In Situ Cell Death Detection Kit, Fluorescein

Protocol tips

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	Fix sample in 10% buffered formalin for 24h before using sample. Block samples in 10% donkey serum in 0.1% Triton-X for 30 min at room temperature. Incubate primary antibody overnight at 4 °C and with secondary antibodies for 1 h at room temperature

Protocol tips
Fix sample in 10% buffered formalin for 24h before using sample. Block samples in 10% donkey serum in 0.1% Triton-X for 30 min at room temperature. Incubate primary antibody overnight at 4 °C and with secondary antibodies for 1 h at room temperature

Publication protocol

At the end of the respective culture durations (24 h, 3 d, 5 d, and 7 d) pelleted islets underwent fixation for 24 h in 10% buffered formalin. The pellet of the fixed isolated islets was then pipetted onto a microscope slide with a circular concave divet in the center. The islets were dispersed throughout the agar as the 2% agar solution was added to the slide. The agar was permitted to harden and then the agar disc was dehydrated and processed to wax through a series of alcohol, xylene, and wax baths. Discs once embedded in paraffin were sectioned at 5 µm thickness.49 Hematoxylin and eosin (H&E) stained histo-processed islets were assessed for physical integrity and cell/tissue turnover (mitosis, necrosis, apoptosis) using the Olympus Compound Microscope by a pathologist who was masked to the study.

Apoptosis was also assessed by the Confocal Microscope LSM 510, Zeiss following in situ hybridization terminal deoxynucleotidyl transferase mediated dUTP nick-end labeling (TUNEL) method50 using the In Situ Cell Death Detection Kit (Fluorescein, 1 684 795, Roche Diagnostics Corporation, Roche Applied Science). Pancreatic tumor tissue following therapy was used as the positive control as it has a naturally high apoptotic index. For the negative control the TUNEL reaction mixture contained only label (fluorescein) and for the positive control, the reaction mixture contained label and enzyme.

Endocrine cell composition was assessed by immunohistochemistry (IHC) where the primary antibodies used were: anti-insulin (raised in mouse) and anti-glucagon, anti-pancreatic polypeptide and anti-somatostatin (raised in rabbit), purchased from Zymed Laboratories Inc. The Dako LSAB 2 System Peroxidase (Universal, K0672, K0673, K0675) kit comprised of biotinylated anti-mouse or anti-rabbit with streptavidin conjugated to horseradish peroxidase, developed with 3,3′-diaminobenzidinetetrahydrochloride (Sigma-Aldrich) and counterstained with Harris’ hematoxylin (Sigma-Aldrich).

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