Journal: Cells

Article Title: Correlative Multi-Modal Microscopy: A Novel Pipeline for Optimizing Fluorescence Microscopy Resolutions in Biological Applications

doi: 10.3390/cells12030354

Figure Lengend Snippet: Correlative Confocal-dSTORM Analysis on Image-Cytometry re-localized cells. ( a ) Representative re-localized G1 cells acquired by the correlative Confocal-STORM protocol described in the text. Confocal Z-Stacks were first acquired of the Tubulin-Atto425 and γH2A.X-Alexa488 fluorescence channel, followed by acquisition of the 53BP1-Cy3 and DynLL1-Alexa Fluor647 channels in dSTORM. The dSTORM plane is identified in the Z-Stack, aligned and merged in the plane (merged image in the second row on the left) and then inserted into the Z-stack (3D projection on the right). ( b ) The described correlative microscopy procedure was applied to cells in the different stages of mitosis, isolated according to the image-cytometry analysis. Z-stacks merged with the dSTORM plane of some representative cells are shown. Scale bar: 5 µm.

Article Snippet: Cells were then rinsed 3 times in PBS and incubated for 1 h with donkey anti-Rat IgG (H + L) biotin (a18749, ThermoFisher Scientific, Waltham, MA, USA) and for 45 min at room temperature with streptavidin Atto425 conjugated (S000-51, Rockland Immunochemicals, Pottstown, PA, USA).

Techniques: Cytometry, Fluorescence, Microscopy, Isolation

Journal: Frontiers in Immunology

Article Title: A Standardized Analysis of Tertiary Lymphoid Structures in Human Melanoma: Disease Progression- and Tumor Site-Associated Changes With Germinal Center Alteration

doi: 10.3389/fimmu.2021.675146

Figure Lengend Snippet: Detection of different TLS phenotypes by 7-color multiplex immunohistochemistry in human melanoma tissues. Examples for (A) early TLS: dense CD20 + lymphocyte aggregates with CXCL13 + cells and the presence of some interspersed CD4 + T cells; (B) primary follicular TLS: CD20 + lymphocyte aggregates interspersed with CD4 + T cells and the presence of a CD21 + but CD23 - dendritic network, surrounded by CXCL13 expressing cells; (C) secondary follicular TLS with a BCL6 - germinal center: CD21 + and CD23 + dendritic networks within CD20 + lymphocyte aggregates with interspersed CD4 + T cells, surrounded by CXCL13 expressing cells. No accessory expression of BCL6 in lymphatic cells. Germinal centers are identifiable in routine light microscopy (see Pathology View, bottom left). An example for a BCL6 + secondary follicular TLS is given in Figure 5A . Images for each of the individual markers and their composites (for clarity without DAPI staining) are shown, together with the corresponding Pathology View (respective bottom left). Scale bars represent 100 µm. (D) Spatial annotation of tumor areas. Left: Definition of the invasive tumor front (red, 2) and the respective intra- and extratumoral compartments (brown, 1 and yellow, 3, respectively) in a tissue section of a human melanoma lymph node metastasis. Extratumoral compartments include site-specific and adipose tissue (yellow). Right: Allocation of intra- (brown/black) and extratumoral perimeters (green) of increasing radiuses (1 – max. 6 mm) drawn within and around the invasive tumor front. Counts and area of TLS were referred to the tissue area (yellow) within the respective intra- and extratumoral perimeters. TLS, tertiary lymphoid structures.

Article Snippet: First, each of the following antibodies was established on four-micrometer sections from FFPE tissue of human tonsil: CD20 (mouse monoclonal IgG2a, clone L26, 1:2000, Agilent, M0755), CD4 (mouse monoclonal IgG1, clone 4B12, 1:500, Agilent, M7310), CXCL13 (rabbit polyclonal IgG, 1:1000, Proteintech, 10927-1-AP), CD21 (rabbit polyclonal IgG, 1:3600, Proteintech, 24374-1-AP), CD23 (rabbit monoclonal IgG, clone SP23, 1:900, Novus Biologicals, NB120-16702) and BCL6 (mouse monoclonal IgG1, clone 1E6B1, 1:24000, Proteintech, 66340-1-Ig).

Techniques: Multiplex Assay, Immunohistochemistry, Expressing, Light Microscopy, Staining

Journal: Frontiers in Immunology

Article Title: A Standardized Analysis of Tertiary Lymphoid Structures in Human Melanoma: Disease Progression- and Tumor Site-Associated Changes With Germinal Center Alteration

doi: 10.3389/fimmu.2021.675146

Figure Lengend Snippet: Metastatic tumor sites: TLS density and spatial distribution. (A) Densities of extratumoral secondary follicular TLS (left) and BCL6 - germinal centers (GC BCL6 - , right) in distant metastatic lymph node and skin sites. (B) Densities of secondary follicular TLS (left) and BCL6 - germinal centers (right) over intra- and extratumoral compartments in distant metastatic lymph node and skin sites. In all boxplots, lower and upper hinges correspond to the first and third quartiles, center line to the median. Upper and lower whisker extend from the hinge to the largest value no further than 1.5 times the interquartile range. Values outside this range are shown as outliers (black circles). Individual patient values are shown as black dots.

Article Snippet: First, each of the following antibodies was established on four-micrometer sections from FFPE tissue of human tonsil: CD20 (mouse monoclonal IgG2a, clone L26, 1:2000, Agilent, M0755), CD4 (mouse monoclonal IgG1, clone 4B12, 1:500, Agilent, M7310), CXCL13 (rabbit polyclonal IgG, 1:1000, Proteintech, 10927-1-AP), CD21 (rabbit polyclonal IgG, 1:3600, Proteintech, 24374-1-AP), CD23 (rabbit monoclonal IgG, clone SP23, 1:900, Novus Biologicals, NB120-16702) and BCL6 (mouse monoclonal IgG1, clone 1E6B1, 1:24000, Proteintech, 66340-1-Ig).

Techniques: Whisker Assay

Journal: Frontiers in Immunology

Article Title: A Standardized Analysis of Tertiary Lymphoid Structures in Human Melanoma: Disease Progression- and Tumor Site-Associated Changes With Germinal Center Alteration

doi: 10.3389/fimmu.2021.675146

Figure Lengend Snippet: Comparison of large BCL6 + secondary follicular TLS in human melanoma to tonsil tissue. (A) Melanoma: CD20 + lymphocyte aggregates with a germinal center surrounded by CXCL13 expressing cells. A CD21 + and CD23 + FDC network covers the complete germinal center area with randomly interspersed CD4 + T cells and BCL6 + cells. No detectable germinal center polarity, no identifiable light and dark zone areas in routine light microscopy (see Pathology View, bottom left). (B) Close up of BCL6 + CD20 + B cells and BCL6 + CD4 + T cells from a BCL6 + secondary follicular TLS in human melanoma. (C) Tonsil: note canonical germinal center polarity with polarized spatial distribution of the CD21 + and CD23 + FDC network together with CD4 + T cells (arrowheads) and a complementary localization of BCL6 + cells (arrow). Identifiable light (arrowhead) and dark zone (arrow) areas in routine light microscopy (see Pathology View, bottom left). (D) Tonsil (upper row): polarized distribution of BCL6 + , Ki67 + (arrows) and BCL6 + Ki67 + cells in germinal centers with canonical germinal center polarity and the presence of light (arrowhead) and dark zone (arrow) areas in routine light microscopy (see Pathology View, left). Melanoma (lower row): note decreased numbers and absence of polarized distribution of BCL6 + , Ki67 + and BCL6 + Ki67 + cells in melanoma germinal centers. Lack of canonical germinal center polarity and absence of light and dark zone areas in routine light microscopy (see Pathology View, left). Images for each of the individual markers and their composites (for clarity without DAPI staining) are shown, together with the corresponding Pathology View (respective (bottom) left). Scale bars represent 100 µm (A, C, D) and 5 µm (B) .

Article Snippet: First, each of the following antibodies was established on four-micrometer sections from FFPE tissue of human tonsil: CD20 (mouse monoclonal IgG2a, clone L26, 1:2000, Agilent, M0755), CD4 (mouse monoclonal IgG1, clone 4B12, 1:500, Agilent, M7310), CXCL13 (rabbit polyclonal IgG, 1:1000, Proteintech, 10927-1-AP), CD21 (rabbit polyclonal IgG, 1:3600, Proteintech, 24374-1-AP), CD23 (rabbit monoclonal IgG, clone SP23, 1:900, Novus Biologicals, NB120-16702) and BCL6 (mouse monoclonal IgG1, clone 1E6B1, 1:24000, Proteintech, 66340-1-Ig).

Techniques: Comparison, Expressing, Light Microscopy, Staining