Citric acid monohydrate

Full formalin-fixed paraffin-embedded (FFPE) tissue sections (3 µm) mounted on glass slides were deparaffinised in xylene and rehydrated in increasing dilutions of ethanol. An antigen retrieval step of 15 min with citric acid monohydrate (pH 6.5; Sigma Aldrich) in a high temperature vapour bath was performed and the endogenous peroxidase activity was quenched with 3% H₂O₂ (VWR International, USA) in methanol (Carl Roth) for 10 min at room temperature (RT). Subsequently, any possible unspecific antibody binding to active Fc receptors was blocked for 1 h at RT using 20% human type AB serum (Sigma Aldrich, Steinheim, Germany, Lot. 017K0443) in phosphate buffer saline (PBS). The slides were then incubated overnight at 4°C with anti-LEF-1 (1:75, Abcam, EPR2029Y), anti-β-catenin (1:2000. TD/BD Biosciences, clone 14) or anti-GR (1:100, Cell signalling, D6H2L) primary antibody (Novus Biologicals) and for negative control, with N-Universal Negative Control Rabbit antibody (Dako). The slides were then washed 5 times with PBS and the primary antibody signal was developed using the HiDef Detection Polymer System Detection Amplifier and HRP Polymer Detector (medac, 954D-20). After counterstaining with haematoxylin, the arrays were then analysed independently by two investigators and different staining intensities were scored semi-quantitatively calculated as followed: (1x percentage of weak staining) + (2x percentage of moderate staining) + (3x percentage of strong staining), ranging from 0 to 300 (34 ). Negative tissue samples were re-analysed in full tissue sections.
The cohort used for immunofluorescence and quantification of immune marker proteins was a sub cohort of the cohort from Landwehr et al. and staining was conducted as described previously (13 (link)). Briefly, the antibodies used were anti-CD3 (1:50, abcam; ab699), anti-CD4 (1:1000, abcam; ab133616), anti-CD8 (1:1000, abcam; ab4055) and anti-FoxP3 (1:40, abcam; ab20034); to investigate the correlation of LEF-1, CTNNB1 and GR expression data.

Betaine, bovine serum albumin (BSA), citric acid monohydrate, erythritol, glycine, hydrochloric acid (HCl), glycolic acid, hydrogen peroxide 30% w/w in water (H₂O₂), isopentyldiol, lactic acid, sodium chloride (NaCl), sodium ethyl laurate sulfate (SLES), sodium hydroxide (NaOH), sodium phosphate dibasic, and monobasic were purchased from Sigma Aldrich. Water used in all protocols was purified by passing through the MilliQ Millipore system.