The slices were stained by routine H.E. staining as well as immunohistochemically stained for AQP1 with the Cell and Tissue staining Rabbit Kit HRP-AEC System (R&D Systems, Minneapolis, MN, USA). The kit was used according to the R&D System’s protocol, and a rabbit primary antibody against AQP1 (Sigma-Aldrich, Merck, Millipore, Burlington, MA, USA) was used at a dilution of 1:400. The samples were than counterstained with hematoxylin (Spitalpharmazie USB, Basel, Switzerland), mounted with aquatex (Merck KGaA, Darmstadt, Germany) and covered up with a Cover-Slip. Every staining was accompanied with a negative control using antibody diluent (DAKO, Glostrup, Denmark).
Immunofluorescence staining was performed as a triple staining with antibodies against AQP1, calretinin, and S100B. The staining was performed according to a standardized protocol, using the rabbit anti-AQP1 antibody (Merck Millipore, Darmstadt, Germany) at a dilution of 1:400, the chicken anti-calretinin antibody (Synaptic Systems, Göttingen, Germany) at a dilution of 1:200 and the guinea pig anti-S100B antibody (Synaptic Systems, Göttingen, Germany) at a dilution of 1:400. As secondary antibodies, anti-rabbit A488 (Invitrogen, Carlsbad, CA, USA), anti-chicken A647 (Invitrogen, Carlsbad, CA, USA), and anti-guinea pig A555 (Invitrogen, Carlsbad, CA, USA) were used at a dilution of 1:2000. Tissue slices were mounted with DAPI (Life Technologies, Thermo Fischer Scientific Inc., Waltham, MA, USA). For every sample an additional negative control without the primary antibodies, was carried out.
Immunofluorescence staining was performed as a triple staining with antibodies against AQP1, calretinin, and S100B. The staining was performed according to a standardized protocol, using the rabbit anti-AQP1 antibody (Merck Millipore, Darmstadt, Germany) at a dilution of 1:400, the chicken anti-calretinin antibody (Synaptic Systems, Göttingen, Germany) at a dilution of 1:200 and the guinea pig anti-S100B antibody (Synaptic Systems, Göttingen, Germany) at a dilution of 1:400. As secondary antibodies, anti-rabbit A488 (Invitrogen, Carlsbad, CA, USA), anti-chicken A647 (Invitrogen, Carlsbad, CA, USA), and anti-guinea pig A555 (Invitrogen, Carlsbad, CA, USA) were used at a dilution of 1:2000. Tissue slices were mounted with DAPI (Life Technologies, Thermo Fischer Scientific Inc., Waltham, MA, USA). For every sample an additional negative control without the primary antibodies, was carried out.
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