Kaiser s glycerol gelatin

Formalin-fixed and paraffin-embedded tumor and spheroid sections (3 µm) were dewaxed using Roticlear (Carl Roth, Karlsruhe, Germany) and rehydrated in a graded series of ethanol. Antigen retrieval was performed in 10 mmol/L citrate buffer pH 6 intermittently heated to 100 °C in 5 min intervals. Washing was performed using 0.05 mol/L Tris-buffered saline pH 8 containing 0.5% (v/v) Tween-20 (TBS-T). Endogenous peroxidase was quenched using 3% H₂O₂ in TBS-T. Endogenous avidin and biotin were blocked using a commercially available avidin/biotin quenching system (Agilent, Santa Clara, CA, USA). Non-specific binding sites were blocked using 10% fetal bovine serum (v/v) in TBS-T. COX-2 was detected using the primary antibody ab15191 (Abcam, Cambridge, UK). Isotype controls were incubated with non-specific rabbit IgG ab27478 (Abcam). Specific binding was detected using the biotinylated secondary antibody 111-065-003 (Dianova, Hamburg, Germany) and ExtrAvidin-peroxidase E2886 (Sigma-Aldrich, St. Louis, MO, USA) followed by staining with 3-amino-9-ethylcarbazole substrate (Sigma-Aldrich). Tumor sections were counterstained with Meyer’s hematoxylin, mounted with Kaiser’s glycerol gelatin (Carl Roth), and imaged using the AXIO Imager A1 microscope (Carl Zeiss, Oberkochen, Germany).

Paraffin sections were dewaxed and hydrated prior to epitope retrieval at pH6. After blocking of endogenous peroxidase (Dako REAL Peroxidase Blocking Solution (Agilent, # S202386-2) sections were incubated with anti-FOXP3 (clone PCH101, ThermoFisher Scientific, 1:50) for 30 minutes at room temperature followed by incubation with secondary antibody (rabbit anti-rat (1:1,000, Dianova # 312-005-003) for 30 minutes at RT. For detection, EnVision+ Single Reagent (HRP. Rabbit) (Agilent # K400311-2) was used. Sections were subjected to epitope retrieval at pH8 prior to incubation with anti-TIRC7 (CellAct, 1:500) for 30 minutes at RT. For detection, the LSAB system was used (Dako REAL Detection System, Alkaline Phosphatase/RED, Rabbit/Mouse (Agilent #K500511-2). Nuclei were stained using Mayer´s hemalaun solution (Merck Millipore # 1092490500) and sections cover-slipped with Kaiser´s glycerol gelatin (Carl Roth GmbH # 6474.1).

For TIRC7 immunohistochemical staining of CCA TMAs, rabbit polyclonal anti-TIRC7 antibody (CellAct, Dortmund, Germany) was used [20 (link),22 (link)]. In brief, 5 µm sections were cut from the formalin-fixed paraffin-embedded samples and deparaffinized and rehydrated according to standard protocols. Sections were then subjected to epitope retrieval at pH8 prior to incubation with anti-TIRC7 antibodies (1:500) for 30 min at room temperature. For detection, the LSAB system was used (Dako REAL Detection System, Agilent Technologies, Santa Clara, CA, USA). For FOXP3 immunohistochemistry, epitope retrieval was performed at pH6. After blocking of endogenous peroxidase (Dako REAL Peroxidase Blocking Solution, Agilent Technologies) sections were incubated with anti-FOXP3 (clone PCH101, 1:50, Thermo Fisher Scientific, Waltham, MA, USA) for 30 min at room temperature followed by incubation with secondary antibody (rabbit anti-rat, 1:1000, Dianova, Hamburg, Germany) for 30 min at room temperature. For detection, EnVision+ Single Reagent (Agilent Technologies, Santa Clara, CA, USA) was used. Nuclei were stained using Mayer’s hemalaun solution (Merck Millipore, Burlington, MA, USA) and sections coverslipped with Kaiser’s glycerol gelatin (Carl Roth GmbH, Karlsruhe, Germany).