Total RNA obtained from different normal tissues (Clontech Laboratories GmbH, Heidelberg, Germany) or from primary tumour biopsies (using RNeasy, Qiagen, Hilden, Germany) was reverse transcribed (SuperScript, Invitrogen, Karlsruhe, Germany) and real-time PCR was performed using the TaqMan system in an AbiPrism 7700 Sequence Detector (Applied Biosystems, Foster City, CA). The oligonucleotides used were (5'-3'):
TCTGTCCTGTTTGCCATATGATGT, CGGAGCAGCCGGACAA, and FAM-AACGTGGA-Amino C6 dT-GAGGGCATCAGCAGCCT (probe).
Transferrin receptor was used to control for RNA integrity, with the following oligonucleotides: GACTTTGGATCGGTTGGTGC, CCAAGAACCGCTTTATCCAGAT, and JOE-TGAATGGCTAGAGGGA-TAMRAdT-ACCTTTCGTCCC (probe)
A standard curve was prepared with synthetic Eag1 RNA. cRNA was prepared using standard protocols  from human Eag1 subcloned into the pSGEM vector (Prof. M. Hollmann, Bochum University). The exact RNA concentration after synthesis was measured by fluorescent labelling (RiboGreen, Invitrogen, Karlsruhe, Germany); different amounts of the synthetic RNA were mixed with total RNA from rat liver; cDNA was prepared from the mixture and processed by TaqMan PCR. The number of PCR cycles required to reach the detection threshold was used to determine tissue mRNA content, using the standard curve for interpolation. Relative amounts of RNA were obtained with respect to the brain Eag1 mRNA content which was set to 1.0. The amplified transferrin receptor was used to normalise the quantity of mRNA from the tissue sample. The constructs used for transient transfection were the already described pTracerCMVhEAG1 and chimera EGFP-hEag1, constructed by introducing an EcoRV site at position 1 of the Eag1 open reading frame (QuickChange Site Directed Mutagenesis Kit, Stratagene, Amsterdam, The Netherlands) and subcloning into the pEGFPC2 vector (Invitrogen, Karlsruhe, Germany).
Antibody design and characterization
A fusion protein containing an area close to the putative pore region (residues 374 to 452, Eag1.62.mAb) and another in the C-terminus of Eag1 (residues 872 to 932, Eag1.33.mAb) was used for immunization. The fusion protein was cloned in the pET32 vector (Novagen, Madison WI, USA), which contains a thioredoxin tag to enhance solubility and a poly-histidine tail that was used for purification after overexpression in E. coli.
Hybridomas were generated by standard techniques (mice immunization, fusion, selection of positive clones and two cloning steps; BioGenes GmbH, Berlin, Germany). The epitope recognised by each supernatant and the ability to differentiate between Eag1 and the most closely related protein known (Eag2 [19–21]) were determined by surface plasmon resonance . Those supernatants with the best performance in this test were then used to examine human brain tissues immunohistochemically and the hybridomas giving the expected staining patterns were subcloned. The antibodies used in this study were subsequently purified by affinity chromatography, first on a protein A column and then on a second affinity column with the fusion protein used for immunization. The activity of the antibody was again checked by surface plasmon resonance prior to use.
To generate a single chain, alkaline phosphatase (PhoA) fused antibody, the antibody cDNA cloning was achieved by first determining the subtype of the murine antibodies (IgG κ2b) using IsoStrip (Roche Applied Science, Mannheim, Germany). Total RNA was prepared from the hybridoma cells (RNeasy kit, Qiagen, Hilden, Germany) and translated into cDNA (SMART PCR cDNA Synthesis Kit, Clontech, Heidelberg, Germany). Subsequently the genes of the light and heavy chains expressed by the hybridomas were amplified by PCR using Pwo polymerase (Roche, Mannheim, Germany) with the following primers:
GTAACAACGCAGAGTACGCGGG and either TCATTTACCCGGAGACCGG (heavy chain) or CTAACACTCATTCCTGTTGAAGCTC (light chain).
The PCR products were subcloned into pBKS+ and sequenced. The variable regions of both chains were identified by sequence comparison  and fused by PCR with a linker sequence between the 3'-end of the heavy chain variable region and the 5'-end of the light chain variable region (Gly-Gly-Gly-Gly-Ser).
Primer sequences were:
TCTGGAGGTGGAGGTAGTGGGGGAGGAGGTTCAGATGTTGTGATGACCCAAACTCC and CCCACTACCTCCACCTCCAGAGCCTCCCCCTCCTGAGGAGACGGTGACTGAGG (heavy chain) and
TCTGGAGGTGGAGGTAGTGGGGGAGGAGGTTCAGATGTTGTGATGACCCAAACTCC and GGCCTAATCGGCCCGTTTGATTTCCAGCTTGGTG (light chain). The fusion was then performed using the first and the last of these sequences.
To produce the PhoA fusion protein, the single chain antibody fragment was amplified and a 5' SalI and a 3' NdeI restriction site introduced. This fragment was cloned (blunt end) into an EcoRV-digested pBKS+ vector prior to subcloning (SalI/NdeI) into pQUANTagen(kx) which contains the alkaline phosphatase sequence. Finally, the single chain antibody fragment – PhoA fusion protein was subcloned into the expression vector pASK-iba2 (using SmaI-SalI sites introduced by PCR)
The fusion protein was obtained from inclusion bodies produced in E. coli (triggered by treatment of anhydro-tetracycline 200 ng/ml). The cells were harvested in 100 mM Tris/HCl, 100 mM NaCl, pH 8.0, 8 M urea and sonicated at 0° until lysis was complete and the solution appeared clear. The lysate was stirred overnight at 4°C and then dialyzed (cut-off 30 kDa) against 6, 4, 2, 1 and 0 M urea in extraction buffer. The solution was then centrifuged at 18000 ×g and stored at 4°C with 0.02 % (w/v) sodium azide. The preparation was characterised by SDS-PAGE, PhoA activity and ELISA to determine the relative activity of the fusion protein compared to the whole murine antibody.
Transfected cells growing on glass coverslips were fixed (4% p-formaldehyde in PBS, 5 min at 4°C), permeabilised with Triton X100 (0.5%, 10 min), blocked (10% BSA, 30 min) and incubated for 2 h with Eag1.62.mAb (1 μg/ml) or Eag1.33.mAb (1 μg/ml). The secondary antibody (AlexaFluor 546 Goat anti-Mouse IgG, Invitrogen, Karlsruhe, Germany) was used at a 1:1000 dilution (30 min) and the preparations were examined by confocal microscopy.
Two postnatal-day 21 Sprague Dawley rats were anesthetised with a mixture of ketamine HCl (Ketaset; 100 mg/ml; Fort Dodge Laboratories, Inc., U.S.A.) and xylazine (Rompun; 20 mg/ml; Mile, Inc., U.S.A.) at 0.1 ml/100 g body weight. The animals were transcardially perfused with 4% p-formaldehyde in 0.12 M phosphate buffer (pH 7.2). After perfusion, the brains were removed, fixed for an additional hour at 4°C, rinsed three times with PBS and stored overnight at 4°C. Coronal and sagittal sections (400–500 μm) were cut in cold PBS using a vibratome (Leica, Vienna, Austria). Slices were incubated for 1 h with 10% normal goat serum in PBS, then with primary hEag1 antibody (Eag1.62.mAb: 2 μg/ml; Eag1.33.mAb: 4 μg/ml) in PBS overnight at 4°C and processed using the avidin biotin-peroxidase system (Vectastain kit, Vector Laboratories, Burlingame, CA). Antibody binding was visualised using 3'-3-diaminobenzidine tetrahydrochloride (DAB; DAB substrate kit for peroxidase, Vector Laboratories). Controls either omitted the primary antibody or incubated the primary antibody with the corresponding fusion protein (10 μg/ml final concentration) at 4°C for 24 h prior to following the procedure as described above. Sections were analysed with a Zeiss Axiophot microscope.
For the immunohistochemical detection of Eag1 potassium channels in human tissues, formalin-fixed and paraffin-embedded biopsy specimens from our own archive, samples from the Manitoba Breast Tumor Bank  and multiple tissue arrays (BioCat, Heidelberg, Germany) were used. Optimal staining conditions and antibody dilutions were determined using formalin-fixed and paraffin-embedded tissue samples from human cerebral cortex. Antigen retrieval was performed in a microwave oven in 10 mM citrate buffer (pH 6.0) at 700 W for at least 15 min. Slides were incubated overnight in a humidified chamber at 4°C with Eag1.62.mAb followed by incubation with the Envision Peroxidase System and DAB (DAKO, Hamburg, Germany). Two operators independently evaluated antigen expression, denoting it either as "negative" (0), "positive" (1) or "strongly positive" (2) as appropriate.
For immunodetection of the single chain antibody fragment alkaline phosphatase fusion proteins, the previous protocol was modified to use the BCIP/NBT (Roche, Mannheim, Germany) substrate and the sample was counterstained with nuclear fast red (DAKO, Hamburg, Germany). Two slides of different multiple tissue arrays were evaluated independently by two researchers using the scoring system described above.
Processing of consecutive sections from the same block in different days resulted in identical staining, illustrating the reproducibility of the system.