Gastric epithelial cell proliferation was studied by BrdU uptake. AGS cells were cultured on 6 well-plates (NUNC) by serum free F-12 medium without antibiotics. Cells were incubated with and/or without H. pylori and IL-16 for 24 h, additionally 1 h incubated with 10 µM BrdU (Sigma-Aldrich) solution. After 3 times wash with PBS, cells were lysed with 50 mM NaCl, 25 mM Tris-HCl, 0.5% NP-40, 0.5% sodium deoxycholate and 0.02% sodium azide. Anti-BrdU (10 µm/ml Dako) and anti-IGF-1R (Abcam) antibodies were diluted with 0.01 M phosphate buffer, 0.15 M NaCl pH 7.2, and coated each well of 96-well plates, and incubated at 4°C overnight. 100 µl of samples were pipetted into wells and incubated for 2 h at room temperature after 3 times washing with Buffer B (0.01 M phosphate buffer, 0.50 M NaCl, 0.1% Tween 20, pH 7.2). The plates were blocked with 3% skim milk (Wako) with Buffer B for 30 min after 3 times washing with Buffer B. The plates were incubated with secondary antibodies (Dako) for 1 h, and further incubated with alkaline phosphatase conjugated streptavidin (Dako) for 1 h. The expressions were followed by ampliQ detection kit (K6245, Dako) and determined by a spectrophotometer at 492 nm.
Alkaline phosphatase conjugated streptavidin
Manufactured by Agilent Technologies
Alkaline phosphatase conjugated streptavidin is a protein complex composed of streptavidin, a protein derived from the bacterium Streptomyces avidinii, and alkaline phosphatase, an enzyme that catalyzes the hydrolysis of phosphate groups. This complex is often used in various bioanalytical techniques, where the high affinity of streptavidin for biotin is utilized to detect and quantify biomolecules of interest.
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Lab products found in correlation
Skim milk, by Fujifilm (1 mentions)
Anti igf1r, by Abcam (1 mentions)
Bromodeoxyuridine (brdu), by Merck Group (1 mentions)
Anti brdu, by Agilent Technologies (1 mentions)
Moma 2, by Santa Cruz Biotechnology (1 mentions)
Alexa fluor 555, by Cell Signaling Technology (1 mentions)
Alexafluor488 anti rat, by Cell Signaling Technology (1 mentions)
2 protocols using alkaline phosphatase conjugated streptavidin
1
BrdU Uptake Assay for Gastric Cell Proliferation
2
Immunohistochemical Staining of Macrophages and Proliferation
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Immunohistochemistry (IHC) was essentially as we described in refs. [15 (link), 16 (link)]. Briefly, sections were fixed in acetone and processed for immunostaining for MOMA-2 (#sc-59332, Santa Cruz Biotechnology, TX) followed by incubation with biotinylated rabbit anti-rat antibody (Dako). After treatment with secondary antibodies sections were incubated with alkaline phosphatase-conjugated streptavidin (Dako). Counterstaining was with hematoxylin. Negative controls were performed by substituting the primary antibody with non-immune IgG from the same species and at the same concentration. Under these conditions, nonspecific immunostaining was not detected. Stained areas were captured (Micropublisher 3.3 Megapixel Cooled CCD Color Digital Camera) and measured (NIS Elements D). Either MOMA-2 or the rabbit polyclonal antibody to AIA31240 (Accurate Chemical and Science Corp.) was used to identify macrophages in co-localization immunofluorescence staining. Secondary antibody was Alexa Fluor-488 goat anti-rabbit (#A11008, Invitrogen, for AIA31240) or Alexa Fluor-488 anti-rat (#4416, Cell Signaling, for MOMA-2). Immunostaining for the Ki67 antigen was performed by incubating acetone-fixed frozen sections with an anti-Ki67 antibody (#ab66155, Abcam; 1:100 dilution) overnight at 4°C followed by anti-rabbit IgG Alexa Fluor-555 (#4413, Cell Signaling) at 1:1,000 dilution for 1 h.
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