rabbit anti na k atpase  (R&D Systems)

Journal: Theranostics

Article Title: Fibroblasts facilitate lymphatic vessel formation in transplanted heart

doi: 10.7150/thno.92103

Figure Lengend Snippet: Fibroblasts Display Activated Phenotype in Allograft Heart. A , t-SNE plot of fibroblast populations of control group ( BALB/c ) and allograft-2 w group. Cd34.high.FB, high expression of FB; ECM.FB, enriched genes and functions related to ECM organization; JF.high.FB, high expression of Jun and Fos ; IFN.FB, high expression of genes related to interferon. B , Pie plot showing the percentage of cell composition in two groups (inside: control; outside: allo-2 w). C , Dot plot shows selected GO terms enriched in fibroblast sub-clusters. D , Violin plot showing the combination expression level (right) of selected activated fibroblast genes (left) in sub-population from two groups. E , Bar plot showing the percentage of activated fibroblasts and other fibroblasts in two datasets. The activated fibroblasts were identified according to combination expression level of genes in Figure d. F , Ridgeline chart showing the expression score of fibrosis-related score in fibroblasts from two groups. G , mRNA quantification of activated fibroblast-associated genes in the hearts from two groups, mRNA expression in the control group as the criterion (1.0). Number of mice in each group: n = 9 (Control), n = 6 (Allo-2 w); unpaired t-test. Postn (Periostin), Col1a1 (Collagen, type I, alpha 1), Col3a1 (Collagen, type III, alpha 1), Ddr2 (Discoidin domain-containing receptor 2), Fn1 (Fibronectin), Pdgfra (Platelet-derived growth factor receptorα). H , I , Immunostaining ( H ) and quantification ( I ) of POSTN expression in control group and allograft group, the area of POSTN expression in the control group as the criterion (1.0). The images within the dashed box are displayed in the second row. Number of mice in each group: n = 4 (Control), n = 4 (Allo-1 w), n = 7 (Allo-2 w); one-way ANOVA test. J , Immuno-staining of POSTN and LYVE1, Collagen I and LYVE1, DDR2 and LYVE1 in two groups. The images within the dashed box are displayed in the second row. Scale bars, 100 µm (H,J). Allo: allograft; FB: fibroblast.

Article Snippet: Primary antibodies were applied in this study as listed: LYVE1 (abcam, ab14917, 1:300), VEGFR3 (R&D Systems, AF349, 1:50), tdTomato (Rockland, 600-401-379, 1:500), Periostin (R&D system, AF2955, 1:50), Collagen Type I (Proteintech, 67288-1-Ig, 1:100 ), DDR2 (discoidin domain receptor 2, R&D Systems, MAB25381, 1:100), PDGFRα (R&D Systems, AF1062, 1:50), PCNA (Abclonal, A0264, 1:500), KI67 (abcam, ab16667, 1:500), VEGFD (Proteintech, 26915-1-AP, 1:50), Midkine (Proteintech, 11009-1-AP, 1:50), SEMA3C (R&D Systems,MAB1728, 1:50), PTN (abcam, ab79411, 1:50), NRP2 (Neuropilin-2, CST, D39A5, 1:50), NCL (nucleolin, Santa cruz, sc-8031, 1:50), Vimentin (abcam, ab8978, 1:100), Fibronectin (abcam, ab2413, 1:50), CD31 (R&D, AF3628, 1:50), CD31 (abcam, ab28364, 1:50), SMA-FITC (Sigma, F3777, 1:300), p-AKT(Santa cruz, sc-514032, 1:50), p-ERK (Santa cruz, sc-7383, 1:50), CD45 (R&D Systems, AF114, 1:50), TNF-α(Proteintech, 60291-1-Ig, 1:50), IL1β (Abclonal, a19635, 1:50), IL6 (Proteintech, 66146-1-Ig, 1:50).

Techniques: Control, Expressing, Derivative Assay, Immunostaining

Journal: Purinergic Signalling

Article Title: Blockage of the adenosine A 2B receptor prevents cardiac fibroblasts overgrowth in rats with pulmonary arterial hypertension

doi: 10.1007/s11302-023-09952-z

Figure Lengend Snippet: Immunophenotypic characterization of cardiac fibroblasts (CFs) isolated from the RV of CTRL and MCT-treated rats on culture day 7. Confocal microscopy images show that CFs exhibit positive immunoreactivity against vimentin (a reliable fibroblast marker; green, A ), α-smooth muscle actin (α-SMA, a myofilament protein typically expressed by activated myofibroblasts; green, D ), and discoidin domain receptor 2 (DDR2, a collagen-specific receptor tyrosine kinase specifically expressed in CFs and myofibroblasts; red, B and E ). Panels C and F show a merge of vimentin and DDR2, and α-SMA and DDR2, respectively. Nuclei are stained in blue with DAPI; yellow denotes co-localization. Scale bar: 30 μm

Article Snippet: Cultured cells were fixed in 4% PFA in PBS for 10 min, washed 3 times in PBS (10 min each), and, subsequently, incubated with blocking buffer I (10% FBS, 1% BSA, 0.1% Triton X, 0.05% NaN 3 ) for 1 h. Primary antibodies diluted in blocking buffer II (5% FBS, 1% BSA, 0.1% Triton X, 0.05% NaN 3 ) were applied [mouse anti-porcine vimentin 1:250 (DAKO); goat anti-human DDR2 1:25 (Santa Cruz); mouse anti-human α-smooth muscle actin (SMA)-FITC 1:250 (Sigma); rabbit anti-human A 2B AR (2nd extracellular loop, 36 kDa) 1:50 (#AAR-003, Alomone Labs)] and the slides incubated overnight at 4 °C.

Techniques: Isolation, Confocal Microscopy, Marker, Staining