Alexa fluor 488 conjugated anti mouse igg antibody

Isolated rat stomach was turned inside out, and it was treated with a Ca₂₊-free DMEM containing 20 mM HEPES (pH 7.4), 0.5% BSA, 0.1 mg/ml trypsin inhibitor and 1 mg/ml Pronase E for 20 min at 37 °C. After the cell debris was removed, the stomach was further digested in DMEM containing 20 mM HEPES (pH 7.4), 0.5% BSA, 0.1 mg/ml trypsin inhibitor, 1.5 mM Pronase E, and 1 mg/ml collagenase for 30 min at 37 °C. The mixture was then filtered through a cell strainer and washed with DMEM. Isolated cells were incubated with 1 mg/ml amphotericin B for 15 min at 37 °C. After washing, the cells were loaded onto a discontinuous Optiprep (Axis-Shield) gradient. After centrifugation at 800 × g for 8 min, the fraction enriched in parietal cells (around 70% of the total) was collected and plated onto collagen-coated coverslips in 12-well plates and incubated at 37 °C in DMEM containing 2 mg/ml BSA, 10 mM glucose, 5 mg/ml ITES, 8 nM EGF, 5 mg/ml G418, 400 mg/ml gentamicin, 20 mg/ml novobiocin, 10 nM hydrocortisone, 100 U/ml penicillin/streptomycin and 20 mM HEPES (pH 7.4). In the experiment using the nanomembrane pH probe sensor, parietal cells were identified by using anti-H⁺, K⁺-ATPase β-subunit antibody (D032-3H, 2B6, Medical & Biological Laboratories) and diluted 1:250 Alexa Fluor 488-conjugated anti-mouse IgG antibody (ab150105, Abcam).

Confocal microscopy was performed with a LSM 700 laser scanning confocal microscope (Carl Zeiss; Jena, Germany). After blocking with 5% BSA solution for 1h, sections were incubated at 4°C overnight with the following primary antibodies: anti-MRP8+MRP14 (1:200, abcam, Cambridge, UK), anti-CD15 (1:200, Bioss, Woburn, MA, USA), and anti CD-68 (1:200, abcam, Cambridge, UK) antibodies. A second layer of Alexa Fluor 488-conjugated anti-mouse IgG antibody (1:1000, abcam, Cambridge, UK) and TRITC-conjugated anti-rabbit IgG antibody (1:1000, abcam, Cambridge, UK) were used as secondary antibodies. Sections were counterstained with 4’-6-diamidino-2-phenylindole (DAPI; Vector, Burlingame, CA, USA).