Fresh frozen tissue slides were air dried, blocked with BB and incubated with BaxB01 (4 μg/ml, in PDAB) for oxMIF detection. The specimen was fixed in 10% PBS buffered formalin (VWR). Furthermore, the sections were incubated with monoclonal mouse anti-Cytokeratin 20 (Dako M7019, 1:50) or anti-CD31 antibodies (e-bioscience 13-0319, 1:200), and monoclonal rabbit anti-BaxB01 antibodies (0.5 μg/ml) diluted in PADB+0.25% TritonX-100. Rabbit and mouse antibodies were detected by Alexa Fluor® conjugated secondary antibodies (Life Technologies A11034 and A21424, 1:2000) diluted in PADB+0.25% TritonX-100. The slides were rinsed in PBS and coverslips were mounted with ProLong Gold Antifade reagent with DAPI (Life Technologies). Pictures were taken at 20-fold magnification (LUCPlanFLN 20x, NA 0.45) with an Olympus inverted microscope (VS81) using a mercury lamp and the filters DAPI (U-MWU2), FITC (U-MWIBA3) and TRITC (U-MWIGA3), and a XM10 camera (Olympus).
Anti cytokeratin 20
Manufactured by Agilent Technologies
Anti-Cytokeratin 20 is a laboratory reagent used to detect the presence of cytokeratin 20 protein in biological samples. Cytokeratin 20 is a marker expressed in certain cell types and can be used for identification and characterization purposes in research and diagnostic applications.
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Lab products found in correlation
Anti cd31 antibody, by Thermo Fisher Scientific (1 mentions)
Pbs buffered formalin, by Avantor (1 mentions)
Prolong gold antifade reagent with dapi, by Thermo Fisher Scientific (1 mentions)
Alexa fluor conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Xm10 camera, by Olympus (1 mentions)
Alexa fluor 488 goat anti mouse, by Jackson ImmunoResearch (1 mentions)
Alexa fluor 488 donkey anti goat, by Jackson ImmunoResearch (1 mentions)
Donkey serum, by Merck Group (1 mentions)
Neutralized formalin, by Merck Group (1 mentions)
Donkey anti rabbit alexa fluor 647, by Jackson ImmunoResearch (1 mentions)
Fluoromount, by Zeiss (1 mentions)
Anti ki67, by Abcam (1 mentions)
Anti fibronectin, by Merck Group (1 mentions)
Axio observer 7, by Zeiss (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Triton x 100, by Merck Group (1 mentions)
2 protocols using anti cytokeratin 20
1
Immunofluorescence Staining of Tissue Slides
2
Immunofluorescence Staining of Cell Cultures
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Cells were fixed with 10% neutralized formalin (Sigma), permeabilized with 0.5% Triton X-100 (Sigma), and blocked for at least 2 h with a blocking buffer containing 1% BSA (Sigma), 3% donkey serum (Millipore), and 0.2% Triton X-100 in PBS. The primary antibodies used were: antifibronectin (1:300, Sigma), anti-Ki67 (1:100, abcam), anticytokeratin 20 (1:100, Dako), anti-myoD monoclonal antibody (MYOD) (2 µg/ml, Sigma) and anti-Myosin Heavy Chain antibody (MYH1E) (1:5, DSHB). All samples were incubated with the primary antibodies overnight at 4°C, followed by a 1 h incubation at RT with secondary antibodies Alexa Fluor® 488 donkey anti-goat, Alexa Fluor® 488 goat anti-mouse, and Alexa Fluor® 647 donkey anti-rabbit (Jackson ImmunoResearch) diluted at 1:500. Alexa Fluor® 568 phalloidin was used to stain filamentous actin (F-actin). Nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI) (1:1000). Finally, samples were mounted with Fluoromount® and observed using an Axio Observer 7 (Carl Zeiss).
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