So far, we were able to show, that the anti-Kv10.2 antibody only labeled neurons in the investigated regions of the mouse brain. To further localize the cellular localization of Kv10.2, we performed chromogenic IHC analysis of lung and liver sections of the adult mouse. As shown in Figure 33, we found only a weak staining of lung epithelial cells. Interestingly, staining of cilia-like protrusions of the cells was observed (Figure 33A, arrows). The structure recognized by the anti-Kv10.2 antibody had a high similarity with the structures detected when applying an anti-acetylated tubulin antibody in IHC (Figure 33C, arrows). The anti-acetylated tubulin is reported marker, besides others, of cilia and the primary cilium of every cell (Alieva et al., 1999).
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Figure 33: Anti-Kv10.2 antibody recognizes cilia-like cellular protrusion in epithelial cells of mouse lung by IHC analysis
A: Chromogenic IHC analysis of mouse lung sections performed with anti-Kv10.2 antibody (NBT/BCIP, blue) resulted in a staining of epithelial cells and of a cilia-like structure (red arrows). B: Negative control, consisting of the HRP conjugated secondary anti-rabbit antibody and the detection system, showed no staining. C: IHC analysis of mouse lung sections performed with anti-acetylated tubulin antibody (NBT/BCIP, blue) resulted in a staining of epithelial cells and a cilia-like structure similar to the structure detected by anti-Kv10.2 antibody (red arrows). D: Negative control, consisting of the secondary HRP conjugated anti-mouse antibody and the detection system, showed no staining. A-D: Sections were counterstained with NFR. Paraffin sections obtained at 7 µm. Scale bar represents 10 µm.
106 After detection of this cilia-like structure in lung sections with the anti-Kv10.2 antibody, we wanted to perform further IHC analysis to investigate if the anti-Kv10.2 antibody also recognizes the cilia of cells of different tissue. Hence, we analyzed liver sections by IHC. Cells of the liver only have one cilium, which is the primary cilium. Again, we observed staining of cilia-like protrusions of the cells (Figure 34A, arrows), which were similar to the staining obtained when using the anti-acetylated tubulin antibody (Figure 34C, arrows). Therefore, this finding suggests, that the anti-Kv10.2 antibody might stain in IHC analysis a structure corresponding to the primary cilium.
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Figure 34: Anti-Kv10.2 antibody recognizes cilia-like cellular protrusion in mouse liver by IHC analysis A: Chromogenic IHC analysis of mouse liver sections performed with anti-Kv10.2 antibody (NBT/BCIP, blue) resulted in a staining of a cilia-like structure (arrows). B: Negative control, consisting of the secondary HRP conjugated anti-rabbit antibody and the detection system, showed no staining. C:
Chromogenic IHC analysis of mouse liver sections performed with anti-acetylated tubulin antibody (NBT/BCIP, blue) resulted in a staining of a cilia-like structure similar to the structure detected by anti-Kv10.2 antibody (arrows). D: Negative control, consisting of the secondary HRP conjugated anti-mouse antibody and the detection system, showed no staining. A-D: Sections were counterstained with NFR.
Paraffin sections obtained at 7 µm. Scale bar represents 10 µm.
Further chromogenic IHC analysis on mouse brain sections resulted in stained cilia-like protrusions of the cells in the mouse cortex and cerebellum (Figure 35A,C, arrows). These cilia- like structures were highly similar with the structures we detected when applying the antibody
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Figure 35: Anti-Kv10.2 antibody recognizes cilia-like structures in mouse cortex and cerebellum by IHC analysis
A: Chromogenic IHC analysis of mouse cortical section performed with anti-Kv10.2 antibody (NBT/BCIP, blue) resulted in a staining of a cilia-like structure (arrows). B: IHC analysis of the cerebellum of mouse performed with anti-Kv10.2 antibody (NBT/BCIP, blue) resulted in a staining of a cilia-like structure (red
108 arrows). C: IHC analysis of mouse cortical section performed with anti-adenylyl cyclase III (ACIII) antibody (NBT/BCIP, blue) resulted in a staining of a cilia-like structure similar to the structure detected by anti-Kv10.2 antibody (arrows). D: IHC analysis of mouse cortical section performed with anti-ACIII antibody (NBT/BCIP, blue) resulted in a staining of a cilia-like structure similar to the structure detected by anti-Kv10.2 antibody (arrows). E: Negative control of cortex section, consisting of the secondary HRP conjugated anti-rabbit antibody and the detection system, showed no staining. F: Negative control of the cerebellum, consisting of the secondary HRP conjugated anti-rabbit antibody and the detection system, showed no staining. A-F: Sections were counterstained with NFR. Paraffin sections obtained at 7 µm. Scale bar represents 10 µm.
anti-adenylyl cyclase III (ACIII) in IHC analysis of the same regions (Figure 35C,D arrows). ACIII is a known marker for primary cilia of the cells of the brain (Bishop et al., 2007). Comparing the staining pattern obtained by the antibody against acetylated tubulin and ACIII with the staining obtained by our antibody we found that the anti-Kv10.2 antibody recognizes a similar structure. This probably suggests a potential localization of Kv10.2 in cilia and to the primary cilium. However, this finding obviously needs to be analyzed in more detail in further studies.
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5 Discussion
The aim of the present study was to analyze the Kv10.2 protein expression pattern in mice, to localize Kv10.2 in different cells of the CNS and to unravel Kv10.2 temporal expression during embryogenesis. Due to a lack of commercially available antibodies specific to Kv10.2, we generated and evaluated a novel polyclonal antibody directed against Kv10.2. The expression pattern of Kv10.2 in mouse tissue detected with the novel anti-Kv10.2 antibody in Western blot revealed a virtually ubiquitious Kv10.2 distribution. Studies of Kv10.2 distribution in the mouse brain with this antibody by IHC showed an expression of Kv10.2 in specific brain regions and indicated localization to neurons. Furthermore, the novel antibody anti-Kv10.2 detected a cellular structure similar to a cilium on mouse tissue such as brain, lung and liver.