Citrate buffer

The protein localization in tissues was assessed using immunohistochemistry as described [42 (link)]. In short, formalin fixed, paraffin-embedded tissue specimens were cut in 3 µm thick sections. The slides were boiled in a microwave oven twice (2 × 10 min, 200 W) in citrate buffer (pH 6.0, 0.1 mM citric acid solution, 0.1 mM sodium citrate solution; Avantor Performance Materials Poland S.A., Gliwice, Poland) and then cooled at RT. In order to block endogenous peroxidase activity, slides were incubated for 3 min in 3% hydroxyperoxide solution (Avantor Performance Materials Poland S.A., Gliwice, Poland). To avoid nonspecific antibody binding, the slides were placed in TBS-T buffer (containing 3% BSA) for 1 h at RT. IHC reactions were performed with the use of the previously described primary antibodies at a 1:100 dilution. Reactions were visualized through subsequent incubation with secondary biotinylated goat anti-rabbit immunoglobulins and 3,3′-diaminobenzidine chromogen (K3468, Dako, Glostrup, Denmark). The slides were counterstained with hematoxylin. The assessment was performed using light microscopy via the Olympus CX41 microscope (Olympus Corporation, Tokyo, Japan).

Protein localization was assessed using standard immunocytochemical procedures as described previously [57 (link)]. Formalin-fixed cell smears were prepared and boiled in a microwave oven twice (2 × 10 min, 200 W, water bath) in citrate buffer (pH 6.0, 0.1 mM citric acid solution, 0.1 mM sodium citrate solution; Avantor Performance Materials; Poznan, Poland). After each step of microwaving antigen retrieval, slides were chilled (20 min, room temperature) and incubated for 3 min in a 3% hydrogen peroxide solution (Avantor Performance Materials, Poznan, Poland). The next step included placing the slides in TBS-T buffer (containing 3% bovine serum albumin, fraction V; Merck KGaA; Darmstadt, Germany) for 1 h at RT. The same primary antibodies were used as described in the western blot section at a 1:100 dilution for all immunocytochemical reactions. Unbounded antibodies were washed in TBS-T buffer (3 × , 10 min, RT). The antigens were visualized using EnVision + /HRP system and 3,3′-diaminobenzidine chromogen (Agilent Dako, Santa Clara, USA). Controls included detection reactions carried out under identical conditions, except that the primary antibodies were replaced by nonimmune serum. The slides were assessed using light microscopy (Olympus CX41 microscope, Olympus Corporation, Tokyo, Japan).