Sagittal sections through E15 and adult rat brain including lateral choroid plexus were selected from the collection of rat tissue at the Faculty of Health and Medical Sciences, University of Copenhagen and used for immunohistochemical detection of SLC16a10, Slc38a5 (SNAT5), Slc4a1, Slc11a1 (NRAMP), Slc39a4 (ZIP4), and Slc1a3 (EAAT1). Sections were deparaffinised in xylene, rehydrated through graded alcohols followed by treatments in 0.5% hydrogen peroxide in methanol for 15 min and rinsing in TRIS buffered saline (TBS) as described previously (Liddelow et al., 2012 (link)). Following removal of non-specific binding by incubation for 30 min with blocking buffer (ChemMate antibody diluent S2022, DakoCytomation, Glostrup, Denmark) at room temperature sections were incubated in primary antibodies as listed in Table 1 .
After overnight incubation sections were washed in TBS and incubated for 30 min in EnVisionTM+ System/HRP (DAKO), K5007, for rabbit antibodies and RPN1025 from GE Healthcare for donkey anti-goat antibodies and then Vector: Vectastain RTU elite ABC reagent PK 7100. This was followed by 6 min incubation with DAB-chromogen solution (DAKO) and counterstaining with Mayer's haematoxylin, dehydrated and mounted with DPX. Control sections contained no primary antibodies and were always blank.
After overnight incubation sections were washed in TBS and incubated for 30 min in EnVisionTM+ System/HRP (DAKO), K5007, for rabbit antibodies and RPN1025 from GE Healthcare for donkey anti-goat antibodies and then Vector: Vectastain RTU elite ABC reagent PK 7100. This was followed by 6 min incubation with DAB-chromogen solution (DAKO) and counterstaining with Mayer's haematoxylin, dehydrated and mounted with DPX. Control sections contained no primary antibodies and were always blank.
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