Rabbit anti chromogranin a

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.

The SI and colon sections were fixed in 4% paraformaldehyde and embedded in paraffin. For histological analysis, tissues were stained with hematoxylin and eosin or Periodic acid–Schiff. For immunofluorescence staining, sections were deparaffinized with xylene and rehydrated in a series of alcohol baths. Heat-induced antigen retrieval was performed in sodium citrate buffer (10 mM, pH 6.0) at 97°C for 10 minutes. After cooling, tissue sections were permeabilized with 0.5% Triton X-100 for 10 minutes, and blocked with 5% bovine serum albumin in phosphate-buffered saline (PBS) for 1 hour, followed by staining with primary antibodies overnight at 4°C. Slides were washed in PBS, stained with secondary antibodies at room temperature for 1 hour, and then incubated with 4′, 6-diamidino-2-phenylindole (Thermo Fisher Scientific, Wilmington, DE) for 2 minutes at room temperature, before mounting with PermaFluor mounting medium (Thermo Fisher Scientific). After staining, images were captured on an LSM 710 confocal microscope (Carl Zeiss, Oberkochen, Germany). Primary antibodies were rabbit anti-Lysozyme (clone EPR2994(2), Abcam, Cambridge, UK), rabbit anti-Chromogranin A (Abcam), or rat anti-CD44 (clone IM7, BD Biosciences, Franklin Lakes, NJ). Secondary antibodies were Alexa Fluor 488 donkey anti-rat IgG or Alexa Fluor 546 donkey anti-rabbit IgG (Thermo Fisher Scientific).