Journal: Scientific reports

Article Title: miRNA in situ hybridization in circulating tumor cells--MishCTC.

doi: 10.1038/srep09207

Figure Lengend Snippet: Figure 1 | Schematic illustration of the MishCTC method for simultaneous miRNA and CK detection via immunocytochemistry. (A) Recovery of peripheral blood into an EDTA tube; (B) blood transfer into a density-gradient centrifuge tube; (C) centrifugation at 700 3 g for 30 min; (D) recovery of the interphase layer, which contains mononuclear and tumor cells, and immunomagnetic labeling with magnetic microbeads conjugated to an anti-CK antibody; (E) magnetic cell separation using a MiniMACS separator and a pre-filled separation column; (F) elution of retained cells; (G) application of the cells to a polylysine glass slide using CytoSpin centrifugation; and (H) MishCTC detection of miRNA and CK. Mr. Juan M. Agudo helped to prepare this artwork.

Article Snippet: The samples were then incubated for 15 min in a blocking solution (0.1% Tween, 2% sheep serum and 1% BSA in 13 PBS) followed by incubation for 15 min in a solution containing both a FITC-anti-cytokeratin antibody (clone: CK3-6H5; Miltenyi Biotec) and an anti-DIG alkaline phosphatase antibody (Roche Diagnostics, Germany).

Techniques: Immunocytochemistry, Gradient Centrifugation, Centrifugation, Labeling, Magnetic Cell Separation

Journal: Scientific reports

Article Title: miRNA in situ hybridization in circulating tumor cells--MishCTC.

doi: 10.1038/srep09207

Figure Lengend Snippet: Figure 2 | Image galleries obtained with the MishCTC method. (a) CK and miRNA-21 expression in an MDA-MB468 cell that was spiked into a blood sample from a healthy volunteer. Detection of cytokeratin-positive (CK1) cells (green channel), miRNA-21-positive cells (red channel) and nuclei (blue channel). Epithelial cells were identified in a leukocyte population that did not express miRNA-21. (b) CK and miRNA-21 expression in a CTC from a patient with metastatic lung cancer. All the CTCs that were found within this set of patients were both CK- and miRNA-positive (upper panel). CK expression in a circulating epithelial cell from a cancer-free patient undergoing a nephrectomy. CK protein expression (green) was detected by immunofluorescence, but miRNA-21 could not be detected by in situ hybridization (lower panel).

Article Snippet: The samples were then incubated for 15 min in a blocking solution (0.1% Tween, 2% sheep serum and 1% BSA in 13 PBS) followed by incubation for 15 min in a solution containing both a FITC-anti-cytokeratin antibody (clone: CK3-6H5; Miltenyi Biotec) and an anti-DIG alkaline phosphatase antibody (Roche Diagnostics, Germany).

Techniques: Expressing, Immunofluorescence, In Situ Hybridization

Journal: Cell reports

Article Title: Aging disrupts sympathetic innervation of the thymus

doi: 10.1016/j.celrep.2026.117126

Figure Lengend Snippet: (A) Representative confocal z stack projection of a 50 μm cryosection demonstrating the loss of β3-tubulin + innervation of large blood vessels immediately before entering the thymic medulla (white arrowheads). The abrupt ending of nerves occurred at the same location as a reduction in α-SMA + cells, and the medulla contains non-vascular α-SMA + cells (green arrowhead). (B) TH + sympathetic innervation ends (yellow arrowhead) along a blood vessel that projects from the cortex, labeled with the cTEC marker keratin 8, into the medulla, labeled with the mTEC marker keratin 5. (C and D) TH + /β3-tubulin + sympathetic nerves innervating a large vessel along the cortico-medullary junction, with cortex/medulla denoted by cell density (Hoechst, C) and mTEC marker (keratin 8, D). Representative confocal z stack projection of TH + sympathetic innervation ending along a blood vessel that enters the medulla (blue arrowheads). Dashed white lines represent cortico-medullary junctions. (E and F) Quantification of TH + segments per thymic region for all sections (E) and average per mouse (F); n = 3–4 sections per mouse for 4 mice. Data are presented as mean ± standard error of the mean and analyzed with repeated measures one-way ANOVA with Tukey’s multiple comparisons test. Subcap, subcapsular cortex; Ctx, cortex only; Med, medulla only; CMJ, cortico-medullary junction; and Ctx and Med, both cortex and medullary regions. Scale bars, 100 μm in (A)–(D).

Article Snippet: Cytokeratin 8 (KRT8) Guinea Pig Polyclonal Antibody , Acris Antibodies , Cat#BP5075; RRID: AB_979823.

Techniques: Labeling, Marker

Journal: Journal of tissue engineering and regenerative medicine

Article Title: A Bruch's membrane substitute fabricated from silk fibroin supports the function of retinal pigment epithelial cells in vitro.

doi: 10.1002/term.2089

Figure Lengend Snippet: Figure 2. Morphology of RPE cells (ARPE-19) after 16 weeks of cultivation on fibroin membranes. (A) A cobblestone morphology accompanied by pigmentation (P, labelled by arrows) was evident within 12 weeks; assessment of fixed cultures after 16 weeks revealed a mostly circumferential arrangement of Factin with occasional stress fibres (C; stained with rhodamine phalloidin). Positive immunostaining (B; relative to negative control) for ZO-1 (D), ezrin (E), cytokeratin pair 8/18 (F), RPE-65 (G) and Na+/K+-ATPase (H) was evident. The distribution for ZO-1 staining resembled the circumferential distribution for F-actin, with (C, D) displaying identical fields of cells. The most intense staining for ezrin was observed at the apical cell surface; refer to Z–Y panel at right of (E). Hoechst nuclear dye is displayed as the blue counterstain (B, D–H). Scale bar = 100 μm and applies to all images

Article Snippet: Primary monoclonal antibodies to zona occludens-1 (ZO-1; 1:100, 33-9100, clone ZO1-1A12, Life Technologies), ezrin (1:100, 35-7300, clone 3C12, Life Technologies), cytokeratin pair 8/18 (1:10, DM189, clone 5D3, Acris Antibodies), RPE65 (1:50, ab13826, clone 401.8B11.3D9, Abcam) and sodium potassium ATPase (Na+/K+-ATPase, 1:100, ab7671, clone 464.6, Abcam) were used to highlight functional Copyright © 2015 John Wiley & Sons, Ltd. J Tissue Eng Regen Med 2015.

Techniques: Staining, Immunostaining, Negative Control

Journal: Journal of tissue engineering and regenerative medicine

Article Title: A Bruch's membrane substitute fabricated from silk fibroin supports the function of retinal pigment epithelial cells in vitro.

doi: 10.1002/term.2089

Figure Lengend Snippet: Figure 3. Morphology of RPE cells (ARPE-19) after 16 weeks of cultivation on commercial polyester membranes (Transwell®). (A) A cobblestone morphology accompanied by pigmentation (P, labelled by arrows) was evident within 12 weeks. Assessment of fixed cultures after 16 weeks revealed a mostly circumferential arrangement of F-actin with less evidence of stress fibres (C; stained with rhodamine phalloidin). Positive immunostaining (B; relative to negative control) was again observed for ZO-1 (D), ezrin (E), cytokeratin 8/18 (F), RPE-65 (G) and Na+/K+-ATPase (H). As for cultures grown on fibroin, the distribution for ZO-1 staining resembled the circumferential distribution for F-actin, with (C, D) displaying identical fields of cells. The most intense staining for ezrin was observed at the apical cell surface; refer to Z–Y panel at right of (E). While staining for cytokeratin pair 8/18 varied according to the Z axis within each cell (F), a more homogeneous distribution was observed for RPE-65 and Na+/K+-ATPase (G, H, respectively). Hoechst nuclear dye is displayed as the blue counterstain in (B, D–H). Scale bar = 100 μm and applies to all images

Article Snippet: Primary monoclonal antibodies to zona occludens-1 (ZO-1; 1:100, 33-9100, clone ZO1-1A12, Life Technologies), ezrin (1:100, 35-7300, clone 3C12, Life Technologies), cytokeratin pair 8/18 (1:10, DM189, clone 5D3, Acris Antibodies), RPE65 (1:50, ab13826, clone 401.8B11.3D9, Abcam) and sodium potassium ATPase (Na+/K+-ATPase, 1:100, ab7671, clone 464.6, Abcam) were used to highlight functional Copyright © 2015 John Wiley & Sons, Ltd. J Tissue Eng Regen Med 2015.

Techniques: Staining, Immunostaining, Negative Control