Journal: The Journal of Clinical Investigation

Article Title: CaMK4 compromises podocyte function in autoimmune and nonautoimmune kidney disease

doi: 10.1172/JCI99507

Figure Lengend Snippet: (A) Representative images of immunofluorescence for nephrin or CaMK4 from patients with LN (n = 10) or FSGS (n = 10) or from a subject without renal disease. Green or red color represents nephrin or CaMK4, respectively, and merged figure shows the signals from nephrin, CaMK4, and DAPI staining. Scale bar: 50 μm. (B) Representative images of immunofluorescence for nephrin or CaMK2 from patients with LN (n = 10) or FSGS (n = 10) or from a subject without renal disease. Green or red color represents nephrin or CaMK2, respectively, and the merged figure shows the signals from nephrin, CaMK2, and DAPI staining. Scale bar: 50 μm. (C and D) Time course of CaMK4 expression in human podocytes after exposure to IgG from LN patients (L) or healthy donors (N) (C) or LPS (D). The data are representative of 3 independent experiments. (E and F) Time course of CaMK2 (arrow) expression in human podocytes after exposure to IgG from LN patients or healthy donors (E) or LPS (F). The data are representative of 3 independent experiments.

Article Snippet: Proteins were transferred to a nitrocellulose membrane, which was subsequently blocked for 1 hour using 5% nonfat dry milk or 3% BSA in TBS-T and incubated at 4°C overnight with mouse anti-human/mouse CaMK4 (catalog 610276/clone 26, BD Biosciences), mouse anti-human/mouse CaMK2 (catalog 611293/clone 45, BD Biosciences), goat anti-human/mouse synaptopodin (catalog sc-21537, Santa Cruz Biotechnology Inc.), mouse anti-human GAPDH (catalog 649202/FF26A/F9, BD Biosciences), mouse anti-RhoA (catalog STA-403-A, Cell Biolabs), mouse anti-Rac1 (catalog STA-401-1, Cell Biolabs), sheep anti-TRPC5 (catalog OST00054G, Thermo Fisher), rabbit anti-FLAG (catalog F7425, MilliporeSigma), mouse anti–phospho-Thr196/200 CaMK4 (provided by Naohito Nozaki, Monoclonal Antibody (MAB) Institute Inc., Iida, Japan), mouse anti–phospho-serine (catalog 612546, BD Biosciences), and mouse anti–14-3-3β (catalog sc-25276/A-6, Santa Cruz Biotechnology Inc.) antibodies.

Techniques: Immunofluorescence, Staining, Expressing

Journal: Histochemistry and Cell Biology

Article Title: Multispectral imaging reveals the tissue distribution of tetraspanins in human lymphoid organs

doi: 10.1007/s00418-015-1326-2

Figure Lengend Snippet: Spectral imaging analysis of human spleen stained for CD3 (Warp Red), CD37 (True Blue) and cell nuclei (Nuclear Red). a Representative original multispectral image. Scale bar = 100 μm. Composite RGB image ( b ) of unmixed CD3 ( c in red ), CD37 ( d in blue ) and Nuclear Red ( e in green in composite RGB image) signal using the spectral library (Fig. a). f Tissue segmentation; B cell follicle (B, yellow ), T cell zone (T, red ), red pulp (RP, green ) and other tissue (blood vessels, collagen; blue ). g Segmentation of individual cells ( green ) based on Nuclear Red staining. h Thresholds for Warp Red and True Blue staining were set to score CD3 − CD37 dim ( blue ), CD3 + CD37 dim ( red ), CD3 + CD37 bright ( yellow ) or CD3 − CD37 bright ( green ) cells. i–l Scatter plots showing optical densities for CD3 (Y-axis) and CD37 (X-axis) of individual cells in T cell zones ( i , l green ), B cell follicles ( j , l blue ) and red pulp regions ( k–l red ) and thresholds used for scoring ( dotted lines ). A representative of 2000 cells per tissue region is plotted

Article Snippet: Tissues were blocked with 2 % HS in Tris-buffered saline (TBS) with 1 % BSA for 45 min at RT and stained with primary antibodies against human CD3 (clone CD3-12, AbD Serotec) or CD20 (clone L26, Thermo Scientific) diluted in TBS supplemented with 1 % BSA overnight at 4 °C.

Techniques: Imaging, Staining

Journal: Histochemistry and Cell Biology

Article Title: Multispectral imaging reveals the tissue distribution of tetraspanins in human lymphoid organs

doi: 10.1007/s00418-015-1326-2

Figure Lengend Snippet: Localization and expression of CD37 in bone marrow ( a–f ), spleen ( g–l ) and appendix ( m–r ). a , g , m Representative original multispectral image of lymphoid organ stained for CD3 (Warp Red), CD37 (True Blue) and cell nuclei (Nuclear Red). Scale bars = 100 μm. b , h , n Composite RGB image after spectral unmixing of original image ( red CD3, blue CD37, green nuclei). c , i , o Image showing scoring of CD3 − CD37 dim ( blue ), CD3 + CD37 dim ( red ), CD3 + CD37 bright ( yellow ) or CD3 − CD37 bright ( green ) cells. Optical density of CD37 on individual cells in human bone marrow ( d ), in B cell follicle ( blue line ) and in T cell zone and in red pulp ( red line ) in human spleen ( j ) and in B cell follicle ( blue line ) and in lamina propria ( red line ) in human appendix ( p ). Optical densities were binned per 0.05 and normalized to % of max. Percentage of CD37 dim and CD37 bright cells in total bone marrow ( e ) and in the CD3 + cell population in human bone marrow ( f ). Percentage of total CD37 bright cells in B cell follicle and red pulp ( k ) and in the CD3 + cell population in T cell zone and red pulp ( l ) in human spleen. Percentage of total CD37 bright cells ( q ) and in the CD3 + cell population ( r ) in B cell follicle and lamina propria in human appendix. Each dot represents data of one ×20 image from the lymphoid tissue. The red line represents the mean. * P < 0.05, **** P < 0.0001

Article Snippet: Tissues were blocked with 2 % HS in Tris-buffered saline (TBS) with 1 % BSA for 45 min at RT and stained with primary antibodies against human CD3 (clone CD3-12, AbD Serotec) or CD20 (clone L26, Thermo Scientific) diluted in TBS supplemented with 1 % BSA overnight at 4 °C.

Techniques: Expressing, Staining

Journal: Histochemistry and Cell Biology

Article Title: Multispectral imaging reveals the tissue distribution of tetraspanins in human lymphoid organs

doi: 10.1007/s00418-015-1326-2

Figure Lengend Snippet: Localization and expression of CD53 in bone marrow ( a–f ), spleen ( g–l ) and appendix ( m–r ). a , g , m Representative original multispectral image of lymphoid organ stained for CD20 (Warp Red), CD53 (True Blue) and cell nuclei (Nuclear Red). Scale bars in a , g , m = 100 μm. b , h , n , Composite RGB image after spectral unmixing of original image ( red CD20, blue CD53, green nuclei). c , i , o , Image showing scoring of CD20 − CD53 dim ( blue ), CD20 + CD53 dim ( red ), CD20 + CD53 bright ( yellow ) or CD20 − CD53 bright ( green ) cells. d , j , p , Optical density of CD53 on individual cells in human bone marrow ( d ), in B cell follicle ( blue line ) and in T cell zone and in red pulp ( red line ) in human spleen ( j ) and in B cell follicle ( blue line ) and in lamina propria ( red line ) in human appendix ( p ). Optical densities were binned per 0.05 and normalized to % of max. Percentage of CD53 dim and CD53 bright cells in the CD20 + cell population ( e ) and in the CD3 + cell population ( f ) in human bone marrow. Percentage of CD53 bright cells in the CD20 + cell population in the B cell follicle and red pulp ( k ), and in the CD3 + cell population in the T cell zone and red pulp ( l ) in human spleen. Percentage of CD53 bright cells in the CD20+ cell population ( q ) and in the CD3 + cell population ( r ) in the B cell follicle and lamina propria in human appendix. Each dot represents data of one ×20 image from the lymphoid tissue. The red line represents the mean. * P < 0.05, **** P < 0.0001

Article Snippet: Tissues were blocked with 2 % HS in Tris-buffered saline (TBS) with 1 % BSA for 45 min at RT and stained with primary antibodies against human CD3 (clone CD3-12, AbD Serotec) or CD20 (clone L26, Thermo Scientific) diluted in TBS supplemented with 1 % BSA overnight at 4 °C.

Techniques: Expressing, Staining