Immunohistochemistry for VDR was performed in fixed and paraffin-embedded sections (5 µM) of intestinal resections from damaged mucosa of CD patients or healthy mucosa from colorectal cancer patients [23 (link)].
The heat-mediated antigen retrieval was performed with 10 mM of sodium citrate buffer at pH 6.0 (Dako Target Retrieval Solution) during 20 min at 98 °C. After the inactivation of endogenous peroxidase and blocking the slides during 1 h at room temperature, intestinal tissues were incubated with the primary antibody Anti-VDR antibody (1:200; 12550, Cell Signaling, Danvers, MA, USA) overnight at 4 °C. The samples were incubated for 30 min at room temperature with the Biotinylated Universal Antibody (1:100; BA-1400, Vector, Peterborough, UK) as a secondary antibody. A negative control without the primary antibody incubation was also performed. Afterwards, in order to get a stronger signal, a 30 min room temperature incubation with Vectastain® Universal Elite ABC Kit (Vector) was performed. A signal was developed after 2 min with DAB enhanced substrate (Sigma-Aldrich, Madrid, Spain). All samples were counterstained with hematoxylin, and pictures were obtained with a microscope (Leica DMI 3000).
The heat-mediated antigen retrieval was performed with 10 mM of sodium citrate buffer at pH 6.0 (Dako Target Retrieval Solution) during 20 min at 98 °C. After the inactivation of endogenous peroxidase and blocking the slides during 1 h at room temperature, intestinal tissues were incubated with the primary antibody Anti-VDR antibody (1:200; 12550, Cell Signaling, Danvers, MA, USA) overnight at 4 °C. The samples were incubated for 30 min at room temperature with the Biotinylated Universal Antibody (1:100; BA-1400, Vector, Peterborough, UK) as a secondary antibody. A negative control without the primary antibody incubation was also performed. Afterwards, in order to get a stronger signal, a 30 min room temperature incubation with Vectastain® Universal Elite ABC Kit (Vector) was performed. A signal was developed after 2 min with DAB enhanced substrate (Sigma-Aldrich, Madrid, Spain). All samples were counterstained with hematoxylin, and pictures were obtained with a microscope (Leica DMI 3000).
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