Dab liquid kit

Standard immunohistochemistry was performed to assess the CD44 protein expression in the diverse formalin fixed human skin tissues. Briefly, 5 µg sections of paraffin embedded blocks were deparaffinized and hydrated prior to epitope retrieval treatment with HIER citrate buffer (Zytomed Systems, Berlin, Germany) for 20 min at 95°C. Endogenous peroxidases were blocked by incubation with 1% (V/V) H₂O₂ for 20 min at room temperature, before serum (Agilent, Santa Clara, USA) for 1 h at room temperature and incubated with the primary CD44 antibody overnight at 4°C. Negative controls were incubated solely in TBS-T. After washing steps, the HRP-linked secondary antibody was added. The detection was performed with the DAB liquid kit (Dako, Santa Clara, USA) and finally the reaction was quenched by immersion in tap water. After haematoxylin staining and dehydration, the slices were mounted with Eukitt^® mounting medium (ORSAtec, Bobingen, Germany). All applied antibodies used are listed in the Table 4. The photo documentation was performed with a DMi8 microscope (Leica, Wetzlar, Germany). The CD44s expression was scored by two researchers (J.F. and S.J.B.) as follows: 1 – weak, 2 – medium, and 3 – strong.

Full FFPE slides were deparaffinised and antigen retrieval was performed in 10 mM citric acid monohydrate buffer (pH 6.5) in a pressure cooker for 13 min. Unspecific binding sites were blocked with 20% human AB serum at room temperature (RT) for 1 h and primary antibodies were incubated in an appropriate dilution concentration at RT for 1 h (PRKACA: BD #610980, 1:1000; PRKAR2B: Sigma #HPA008421, 1:40; PRKAR1A: Novus #29250002, 1:250; PRKAR1B: Abnova #H00005575-M05, 1:100; PRKAR2A: BD #612243, 1:5000 and N-Universal Negative Controls anti-rabbit or anti-mouse: Dako). Signal amplification was achieved by En-Vision System Labelled Polymer-HRP Anti-Rabbit (Dako) for 40 min and developed for 10 min with DAB+ Liquid Kit (Dako). Nuclei were counterstained using Mayer’s hematoxylin for 2 min. Specificities of antibodies were determined by WB. All antibodies gave one specific band at the predicted size.