Figure 1.
Specificity of the anti CK1δ polyclonal rabbit serum NC10.
An immunoabsorption test for NC10 was used to show its specificity in immunohistochemistry (IHC). Immersion fixation with acetic formalin, alkaline phosphatase reaction, dye: newfuchsin. IHC was performed on paraffin-embedded pancreatic tissue of a 5 week old BALB/c mouse using NC10 (A) or NC10 preincubated with either a control peptide (the p53 specific peptide MEESQSDISLELGGC, 0.1 µg (B)) or with the specific blocking peptide used for immunisation of rabbits (CGDMASLRLHAARQGARC, 0.1 µg) for 3 h at 4°C (C). The results indicate that the antigenic peptide, but not the control peptide competively inhibits CK1δ binding. Magnification: 400×.
Figure 2.
Immunohistochemical detection of CK1δ in skeletal muscles of a six week old BALB/c mouse.
Longitudinal section. Perfusion fixation with Bouin. Peroxidase reaction, dye: DAB. A similar CK1δ staining pattern of the myofibrils was detected independent of the antibody (NC10, 108, or ab10877). Specific antibody binding was visualised by the peroxidase reaction using DAB as substrate-chromogen. (A) NC10, immersion fixation with acid formalin; (B) 108, perfusion fixation with Bouin; (C): ab10877 (Abcam), immersion fixation with acid formalin. Magnification 100×. Using three different CK1δ specific antibodies only minor variations in the CK1δ staining pattern were observed. These could be explained by alterations in the phosphorylation status of CK1δ influencing the disposability of the particular antigenous epitope and/or by differences in the recognition of CK1δ splice variants being differently expressed in those cases.
Table 1.
Effect of different fixation agents and fixation methods on antigen detection.
Table 2.
Heat induced antigen demasking.
Table 3.
Effect of different blocking reagents on background reduction of CK1δ immunostained frozen sections.
Table 4.
Localization and levels of CK1δ in 4 to 6 week old BALB/c mice.
Figure 3.
Immunohistochemical detection of CK1δ in the gastrointestinal tract, endocrine glands, lung, skin, and mammary gland.
Fixative: acid formalin, fixation by immersion. Peroxidase reaction, dye: DAB. Immunohistochemical staining of CK1δ in the stomach (A); small intestinal (B); colon (C); large salivary gland (D); small salivary gland of the trachea (E); pancreas (F); liver (G); adrenal gland (H); thyroid gland (I); squamous epithelium of upper aerodigestive tract (J); lung (K, L), skin (M), harderian gland (N), and mammary gland (O). In (A), solid arrows point to parietal cells, open arrows to neck parietal cells and open arrowheads to chief cells. In figure 3L, solid arrows point to strong CK1δ positive type II pneumocytes. Magnification: 100× (A, B, K), 200× (G, H, J, N), 400× (C–F, I, L, M, O).
Figure 4.
Immunohistochemical detection of CK1δ in the urogenital tract.
Fixative: acid formalin, fixation by immersion. Peroxidase reaction, dye: DAB. The staining results of the CK1δ specific antiserum NC10 in organs of the urogenital tract are shown. (A, B) kidney; (C) ureter; (D) ovary; (E) fallopian tube; (F) uterus; (G) testis; (H) seminal duct and epididymis; (I) prostate; (J) seminal vesicle. The solid arrow indicates a primary follicle in D. Magnification: 100× (E, F, H), 200× (A, D, I, J), 400× (B, C, G), 640× (A).
Figure 5.
CK1δ expression in immobile cells of mesenchymal origin.
Fixative: acid formalin, fixation by immersion. Peroxidase reaction, dye: DAB. CK1δ specific antiserum: NC10. The CK1δ immunostaining results of striated muscle cells of the skeletal system (A), the myocardium (B), and of smooth muscle cells of the intestinal wall (C); aterial blood vessel (D); venous blood vessel (E); lymphatic vessel (F); mesothelial cells of the peritoneum, (G) mesothelial cells of the pericardium (H), mesothelial cells of the pleura (I), adipocytes of white and brown fatty tissue (J), chondrocytes of the hyaline cartilage (K) and osteocytes (L) are shown. In B, arrows indicate intercalated discs of cardiomyocytes. Magnification: 200× (G, H, J), 400× (A–F, I, K, L).
Figure 6.
Localisation of CK1δ in hematopoetic and lymphoid organs and the eye.
Fixative: acid formalin, fixation by immersion. Peroxidase reaction, dye: DAB. The CK1δ specific antibody NC10 was used to detect CK1δ in hematopoetic and lymphoid organs and in the eye. (A) bone marrow; (B) thymus; (C, D) lymph node; (E, F) cecal lymphoid follicle; (G) the eye ciliary body and iris; (H) lens and iris; (I) retina and lens. Immunohistochemical analysis of frozen retinal sections reveals localisation of CK1δ in retinal ganglion cells, which were specifically co-stained with an anti-βIII-tubulin antibody or DAPI (J–L). Magnification: 100× (B, C, E), 200× (D, F), 400× (A, G–L). ONL: outer nuclear layer; INL inner nuclear layer; GCL: retinal ganglion cell layer. Scale bar: 50 µm. Arrows in D indicate high endothelial venules (HEV), arrows in E delineate a B-follicle. In F closed arrows point to a lymphoid blast, the arrowhead points to a small resting lymphocyte and the open arrow indicates high endothelial venule.
Figure 7.
CK1δ expression in the nervous system.
Fixative: acid formalin, fixation by immersion. Peroxidase reaction, dye: DAB. (A) CK1δ was strongly expressed in the hypophysis. (B) The high-power view shows a cytoplasmatic staining of the epithelial cells of the adenohypophysis (Adeno). The pars intermedia (PI) and the neurohypophysis (N) were also strongly marked. (C, D): The nerve cell perikarya of a spinal (C) and a trigeminal ganglion (D, arrow) exhibit CK1δ whereas the adjacent nerve fibers were not marked. (E) The spinal cord neurons were strongly marked (arrows) whereas the gray matter neuropil and the white matter only faintly exhibited CK1δ (asterix). The inset demonstrates the high power view into the boxed area and shows a marked cytoplasmic labelling. (F) In the hippocampal formation the neurons of the sectors CA1, CA2 and CA3 were strongly labelled (arrows). (G) Sagittal section of a mouse brain immunostained with the NC10 antibody directed against CK1δ. There are especially high levels of CK1δ detectable in all layers of the neocortex (NC), the olfactory bulb (OB), and the molecular and Purkinje-cell layer of the cerebellum (CB). The white matter as seen in the corpus callosum (CC) and the cerebellar white matter did not show high levels of CK1δ. Low levels of CK1δ were found in the thalamus (THAL), midbrain (MB), pons (P) and the medulla oblongata (MO). (H, I) At higher magnification neocortical neurons show a strong cytoplasmatic staining whereas the neuropil was weakly stained. There was no staining of glial cells detectable. (J) In the thalamus there was a very light staining of the neuropil. Some thalamic neurons exhibited CK1δ in the cytoplasm (arrows) whereas other neurons were not labelled (arrowheads). (K) In the cerebellum CK1δ stained the molecular layer (M) and the Purkinje cell layer (P). The granule cell layer neurons (G) were not marked. There was only a very light staining of the neuropil in this layer. (L) At the higher magnification levels it was evident that the CK1δ expression in the molecular layer was the result of the staining of the entire dendritic trees of the Purkinje cells. The arrows indicate a Purkinje cell with the apical dendrite positive for CK1δ. There was no labelling of Bergmann glia cells. (M) In the dentate nucleus of the cerebellum the neuropil was weakly stained whereas some neurons were strongly CK1δ positive (arrows). Neurites were also labelled in this nucleus (arrowheads). Hypothalamic nuclei showed varying levels of CK1δ. Some of these neurons, e.g. in the suprachiasmatic nulceus exhibited nuclear CK1δ. N. supraopticus (N). Calibration bar in L equals: A = 270 µm, B = 120 µm, C, H, L, N = 180 µm, D = 150 µm, E = 400 µm, E-inset = 55 µm F = 280 µm, G = 800 µm, I = 6.6 µm, J, M = 40 µm, K = 70 µm. CK1δ was stained with the antibodies NC10 (A–C, E–N) and abcam 10877 (D).