Rabbit anti active caspase 3

Immunohistochemistry was performed on formalin-fixed paraffin-embedded antral and corpus sections. Antibodies used were rabbit anti-bak (Millipore, Hampshire, UK), rabbit antiactive caspase-3 (R & D Systems, Oxfordshire, UK), rabbit antichromogranin A (Abcam, Cambridge, UK), rabbit anti-H⁺-K⁺-ATPase (Calbiochem, Nottingham, UK), and rat anti-Ki-67 (Dako, Ely, UK). Immunostaining for bak, active caspase-3, chromogranin A, and Ki-67 was performed as previously described (9 (link), 21 (link)). Briefly, antigen retrieval was performed by heat retrieval in 10 mmol/l tricarboxylic acid buffer (pH 6), and the primary antibody was applied at 1:600 for bak, 1:750 for active caspase-3, 1:1,000 for chromogranin A, and 1:20 for Ki-67 overnight at 4°C. A goat antirabbit or rabbit antirat biotinylated secondary antibody (Dako) was used at a dilution of 1:200, and an ABC (Vector Laboratories, Peterborough, UK) amplification step was performed before detection by 3-3′-diaminobenzidine tetrahydrochloride. Immunohistochemistry for H⁺-K⁺-ATPase did not require heat-mediated antigen retrieval, instead, sections were incubated with 1% Triton X-100 for 30 min before the addition of primary antibody at 1:1,000.