For immunohistochemistry, unconjugated polyclonal LgR5 (rabbit), and isotype control antibodies (mouse, rabbit) were purchased from Abcam (Cambrige, UK). The unconjugated mouse monoclonal Cdx-2 antibody was obtained from Biogenex (San Ramon, USA) and the unconjugated mouse monoclonal Ki-67 antibody was purchased from Acris (Hiddenhausen, Germany). The secondary antibody used for immunofluorescence double staining of Ki-67 was a fluoresceinisothiocyanat (FITC)-conjugated AffiniPure donkey-anti-mouse IgG, used at 1:200 dilution (Jackson ImmunoResearch Laboratories Inc.,
Suffolk, England). The secondary antibody check details for LgR5 was a Cy3-conjugated AffiniPure donkey-anti-rabbit IgG (Jackson ImmunoResearch), used at 1:200 this website dilution. Normal colon tissue was used as positive control for LgR5 expression [24, 25]. The colon tissue had undergone the same processing, like the esophageal cancer specimen (normal formalin-fixed, paraffin-embedded tissue from colon resections for benign
conditions – normal colon mucosa adjacent to polyps or diverticular disease). Cell Culture We analyzed LgR5 expression in cells (1 × 104) from the esophageal adenocarcinoma cell line OE-33 (Rabusertib chemical structure Sigma-Aldrich, Steinheim, Germany) in cytospins as additional positive control for LgR5 expression. This cell line is the only commercially available adenocarcinoma cell line of the lower esophagus (Barrett’s metaplasia) and was established from a 73-year-old female patient. The tumor was identified as pathological stage IIA (UICC) and showed poor differentiation. Using RT-PCR we tested negative for mycoplasma contamination of this cell line that was provided to our laboratory in December 2009 by Sigma. The cell line was cultured in RPMI-1640 medium, supplemented with 10% Fetal Bovine Serum, 100 units/ml of penicillin and 100 μg/ml of streptomycin. Cytospins of the OE-33 cell line were fixed in acetone and dried for 10 minutes.
Rehydration, blocking, and the staining procedure steps were the same as described for immunohistochemistry PIK3C2G of FFPE sections. Additionally, RT-PCR was performed for LgR5 gene expression of OE-33 cells. Double Staining Experiments (IF and IHC) The sequential immunofluorescence (IF) double staining (co-expression) was analyzed for LgR5 with Ki-67 expression. Sequential immunohistochemical (IHC) double staining was performed for Cdx-2 and LgR5. Processing of tissue and staining procedure Serial tissue sections (2 μm thickness) were cut from formalin-fixed paraffin-embedded (FFPE) blocks on a microtome and mounted from warm water onto adhesive microscope slides (Hartenstein, Wuerzburg, Germany). Sections were deparaffinized in xylene and ethanol and rehydrated in water. Heat induced epitope retrieval (HIER) was performed with citrate buffer pH 6.0 (Dako, Hamburg, Germany).