Journal: Nature Communications

Article Title: Sfrp5 identifies murine cardiac progenitors for all myocardial structures except for the right ventricle

doi: 10.1038/ncomms14664

Figure Lengend Snippet: ( a ) Representative photographs of LacZ-stained sections from Sfrp5-ERT2Cre / Rosa-lacZ embryos at E10.5, labelled by tamoxifen at E7.5. ( b ) Schematic drawing for the expression of Sfrp5 (orange) and the cardiac contribution of its lineages (red) when cells were labelled at E7.5 and analysed at E10.5. ( c ) Double fluorescent whole-mount ISH of mouse embryos at E7.5 for detection of Sfrp5 (green) and Hcn4 , Tbx5 or Isl1 (red), as indicated in each panel. ( d ) Schematic drawings of gene expression of Sfrp5 , Isl1 , Hcn4 and Tbx5 . ( e ) Double immunohistochemistry of Sfrp5-venusYFP at E8.5 using anti-GFP (green) and anti-HCN4 (red) antibodies. Co-expression with HCN4 was found in the VP. Weak expression of YFP also co-distributed with HCN4-positive cells in the IFT and part of the primitive ventricle (PV). ( f , g ) Double immunohistochemistry of Sfrp5-venusYFP ( e ), Sfrp5-venusYFP/Isl1 - Cre / CAG-(floxed)-CAT-mRFP ( f ) or Sfrp5 - Cre / CAG-(floxed)-CAT-eGFP ( g ) embryos at E9.5 using anti-GFP (green) antibodies and anti-HCN4, anti-RFP, anti - Isl1 or anti-Tbx1 (red) antibodies. Rectangles in upper panels are magnified in lower panels (v: ventral; d: dorsal). Co-expression with Isl1 was detected (arrows in panel ( f ) and parenthesis in panel ( g )). Boundaries for the VP from the atrium are indicated by white dotted lines ( f , g ). Signal for Tbx1, but not GFP, was observed in the anterior splanchnic mesoderm and anterior OFT (arrowheads in panel ( g )). A, atrium.

Article Snippet: After washing with PBS and 1 h of preincubation with 1% BSA in PBS for blocking, sections were incubated with 500 × diluted primary antibodies (anti-GFP: Nakalai GF090R or Abcam ab290; anti-Tbx1: Santa Cruz sc-17874; anti-Tbx18: Santa Cruz SC17869; anti-Wt1: Abcam ab10670; anti-Nkx2-5: Abcam ab35842; anti-TnT: Thermo MS-295-P0; anti-Hcn4: Alomone Labs APC052; anti-RFP: MBL PM005; anti-β-catenin: Millipore 05-665; anti-Ki67: Abcam ab15580) at 4 °C overnight.

Techniques: Staining, Expressing, Gene Expression, Immunohistochemistry, Immunofluorescence

Journal: PLoS ONE

Article Title: Overexpression of Map3k7 activates sinoatrial node-like differentiation in mouse ES-derived cardiomyocytes

doi: 10.1371/journal.pone.0189818

Figure Lengend Snippet: Primer pairs used for real time PCR.

Article Snippet: 1 (Santa Cruz) and anti- Hcn4 (NeuroMab) antibodies.

Techniques:

Journal: PLoS ONE

Article Title: Overexpression of Map3k7 activates sinoatrial node-like differentiation in mouse ES-derived cardiomyocytes

doi: 10.1371/journal.pone.0189818

Figure Lengend Snippet: A. Immunocytochemistry showing that individual or small clusters of cardiomyocytes that form in wild-type and Map3k7 -overexpressing EBs express both the MF20 epitope (anti-CT3 epitope, green) and cardiac Troponin ( cTnI , red). Blue indicates DAPI signal in all panels. Note that Troponin protein is poorly organized in cardiac cells derived from Map3k7 -overexpressing ES cells. Scale bars indicate 10 μM in panels stained for cTnI and 20μM in panels stained for MF20. B. After several weeks in culture Map3k7 -overexpressing cells showed spider and spindle cell morphologies that are characteristic of mature SAN cells. C. Fluorescent image showing Map3k7 -overexpressing cells that also possess the MHCα::mCherry reporter (marking cardiomyocytes with red fluorescence) and wild-type cells possessing the MHCα::GFP reporter (marking cardiomyocytes with green fluorescence). Note that cardiomyocytes derived from Map3k7 -overexpressing cells possess a distinct morphology as compared to wild-type cardiomyocytes. Scale bar represents 100 μM. D. Anti- Map3k7 antibody staining of 19 day old mouse embryo showing increased expression in the remnants of the sinus venous and overlapping with HCN4 positive cells. E. By contrast, low expression of Map3k7 in the left ventricle of the same heart.

Article Snippet: 1 (Santa Cruz) and anti- Hcn4 (NeuroMab) antibodies.

Techniques: Immunocytochemistry, Derivative Assay, Staining, Fluorescence, Expressing

Journal: PLoS ONE

Article Title: Overexpression of Map3k7 activates sinoatrial node-like differentiation in mouse ES-derived cardiomyocytes

doi: 10.1371/journal.pone.0189818

Figure Lengend Snippet: A. Diagram of the major transcriptional regulators of SAN differentiation in the mouse embryo. Tbx5 activates Shox2 , Tbx3 and SAN-specific markers, including Hcn2 and Hcn4 . In addition, Shox2 inhibits Nkx2 .5 expression, and Tbx3 inhibits markers for the working myocardium. B. qRT-PCR data showing changes in the expression or timing of SAN related genes. Blue line indicates relative gene expression normalized to expression of Gapdh over time in wild-type R1 EBs and red lines indicate gene expression in Map3k7 -overexpressing EBs. Error bars indicate standard error from three technical replicates. Upregulation or downregulation of each gene was considered relevant only if relative expression trends were the same in each of a minimum of three biological replicates. Error bars represent standard error from three technical replicates. Statistical significance was determined by t-test. (*) represent p<0.006.

Article Snippet: 1 (Santa Cruz) and anti- Hcn4 (NeuroMab) antibodies.

Techniques: Expressing, Quantitative RT-PCR, Gene Expression

Journal: PLoS ONE

Article Title: Overexpression of Map3k7 activates sinoatrial node-like differentiation in mouse ES-derived cardiomyocytes

doi: 10.1371/journal.pone.0189818

Figure Lengend Snippet: A. Beat Rate Data showing quartile beat rate data for 100 beating foci for two wild type mouse strains and Map3k7 overexpressing cells. B. Immunocytochemistry showing overlap of the cardiac reporter with the SAN specific marker Hcn4 . C. Summary of this data showing the % of Hcn4 expressing cardiomyocytes is dramatically increased in the Map3k7 -overexpressing cells. D. Perforated patch recording of automaticity and pacemaker current in mCherry expressing cardiomyocytes derived from Map3k7 -overexpressing ES cells. Left: Spontaneous APs. Middle: Pacemaker current recorded from the same cell. Right: calculated activation relation from the same cell, showing current activation within the diastolic potential range. E. Calcium transients in cardiomyocytes derived from Map3k7 -overexpressing ES cells before (red trace) and after (blue trace) treatment with either 1μM norepinephrine or 0.01μM acetylcholine, as indicated. Beating in these cardiomyocytes accelerates in response to norepinephrine and slows down in response to acetylcholine.

Article Snippet: 1 (Santa Cruz) and anti- Hcn4 (NeuroMab) antibodies.

Techniques: Immunocytochemistry, Marker, Expressing, Derivative Assay, Activation Assay

Journal: Frontiers in Cardiovascular Medicine

Article Title: Arrhythmogenic Hearts in PKD2 Mutant Mice Are Characterized by Cardiac Fibrosis, Systolic, and Diastolic Dysfunctions

doi: 10.3389/fcvm.2021.772961

Figure Lengend Snippet: Cell number is decreased in Pkd2 -KO mouse atrioventricular node. (A) Heart tissues were stained with anti-HCN4 antibody used as a marker for the myocardial conduction system (green staining). Atrioventricular node (AVN) is identified by both location and HCN4-postive staining indicated by the red circles; RV, right ventricle. (B) The AVN is next identified as region of interest. (C) Number of cell nucleus (DAPI; blue staining) is counted. (D) Data reveal a significantly lower number of cells in AVN of Pkd2 -KO mouse hearts. N = 5 mice for each WT and Pkd2 -KO group at age matched of 6 months old. ** p < 0.01.

Article Snippet: To study fibrotic pathways, anti-β-catenin (1:50; Cat# sc-133240; Santa Cruz, Inc .) and anti-pSMAD3 (1:1,000; Cat# sc-517575; Santa Cruz, Inc ) antibodies were used in the same manner as anti-HCN4 antibody followed with AlexaFluor-488 secondary goat anti-mouse antibody (1:1,000; Cat# ab150113; Abcam, Inc ).

Techniques: Staining, Marker

Journal: Stem Cells International

Article Title: Genetically Modified Porcine Mesenchymal Stem Cells by Lentiviral Tbx18 Create a Biological Pacemaker

doi: 10.1155/2019/3621314

Figure Lengend Snippet: Western blotting and PCR analysis of target protein and mRNA expression level after transfection. (a) Western blotting of Tbx18 expression level after transfection for 72 h. BMSCs without transfection are set as the control. (b) PCR analysis of Tbx18 expression level after transfection for 48 h. ∗ represents P < 0.05. (c) Western blotting detects increased HCN4, α -SA, and cTnI protein expression. (d) Quantitative analysis of the protein expression levels of HCN4, α -SA, and cTnI. ∗ represents P < 0.05. cTnI: cardiac troponin I; GFP: green fluorescent protein; HCN4: hyperpolarization-activated cyclic nucleotide-gated channel 4; α -SA: α -striated actin.

Article Snippet: Primary antibodies against HCN4 (ab32675, 1 : 5000), cTnI (ab47003, 1 : 500), α -SA (ab156302, 1 : 1000), Tbx18 (ab115262, 1 : 800), and GAPDH (Proteintech, 1 : 5000) were incubated overnight at 4°C.

Techniques: Western Blot, Expressing, Transfection, Control

Journal: Molecular Therapy

Article Title: Human ISL1 + Ventricular Progenitors Self-Assemble into an In Vivo Functional Heart Patch and Preserve Cardiac Function Post Infarction

doi: 10.1016/j.ymthe.2018.02.012

Figure Lengend Snippet: In Vitro Differentiation Potential of ISL1 + HVPs (A–D) Day 15+ HVPs were analyzed for ventricular markers: (A) MLC2v and cTnT; (B) cTnI; (C) pacemaker marker HCN4; and (D) atrial marker MLC2a. Scale bar, 20 μm. (E and F) Optical recordings of spontaneous (E) and paced (F) day 18 HVP cells. Isochrome maps at 20-ms intervals show spontaneous action potential initiation. The white asterisk represents the origin of point electrical stimulation. (G and H) Optical recordings of spontaneous (G) and paced (H) calcium transients in day 18 HVP cells. The white asterisk represents the origin of point electrical stimulation. (I and J) Patch clamp recordings of day 15+ (I) and day 39+ (J) HVPs showed typical ventricular-like action potentials. (K) Whole cell patch clamp recordings of day 21 and day 40 HVP-derived ventricular cardiomyocytes. Cultures demonstrated that there were no significant differences in various AP parameters between the two time points, suggesting that in vitro , HVPs reached maturity by day 18. (L) Representative I f traces in the presence of 1 mM Ba 2+ . (M) Patch clamp recording comparison of I f in ES03- and HES2-derived ventricular cardiomyocytes. Steady-state I-V (left) and steady-state activation relations (right) for I f (n = 12). (N and O) Whole cell patch clamp recording of I Na in ES03. (N) Representative I Na traces during activation (left) and inactivation (right). (O) Plots of peak I Na density I-V relationship (left) and steady-state activation/inactivation (right), n = 5. Histograms and traces are shown as mean ± SEM.

Article Snippet: Tissue sections were subsequently stained with various antibodies against αSMA (Thermo Scientific, 14-9760-82, USA); Brachyury (R&D Systems, AF2085, USA); CD31 (Cell Signaling Technology, mab 3528, USA); cTnI (Abcam, ab47003, UK); connexin 43 (Abcam, ab11370, UK); cTnT (Thermo Scientific, MS-295-P1, USA); GFP (Abcam, ab13970, UK); HCN4 (R&D Systems, MAB8138, USA); human fibronectin (R&D Systems, AF1918, USA); ISL1 (DSHB, 39.3F7, USA); Ki67 (BD Pharmingen, 550609, USA); human mitochondria (Millipore, mab1273, USA); laminin (DSHB, 2E8, USA); Mesp1 (Abcam, ab77013, UK); MLC2v (Proteintech, 10906-1-AP); MLC2a (Synaptic Systems, 311-011); MYH11 (Sigma, HPA015310, USA); and VE-cadherin (R&D Systems, USA).

Techniques: In Vitro, Marker, Patch Clamp, Derivative Assay, Comparison, Activation Assay

Journal: Molecular Therapy

Article Title: Human ISL1 + Ventricular Progenitors Self-Assemble into an In Vivo Functional Heart Patch and Preserve Cardiac Function Post Infarction

doi: 10.1016/j.ymthe.2018.02.012

Figure Lengend Snippet: In Vivo Transplantation of HVPs under Renal Capsule (A) Schematic illustration of HVP generation, differentiation, and transplantation. HVPs were injected under the kidney capsule or intra-myocardially into the heart (injection site marked with squares); photos illustrate where the HVP graft was formed 2 months after transplantation, when it was recovered. (B) Electron microscopy of the HVP patch in the kidney capsule revealed mature ultra-structure, including organized and aligned sarcomeres, with well-defined Z-line, M-line, A-band, and I-band (left micrograph). Also apparent were desmosomes, mitochondria, myofilament bundles (middle panel), and capillaries containing red blood cells (right panel). (C) High-resolution immunofluorescence images further demonstrate sarcomeric organization within HVP grafts, with transverse orientation of a-actinin at z-lines and actin staining between z-lines. Membrane staining (WGA) revealed the presence of developing T-tubules (middle panels, high magnification of indicated region at right). (D) Immunofluorescence staining of the HVP kidney patch, with antibodies against MLC2a, MLC2v, cTnT, aSMA, ISL1, laminin, HCN4, human fibronectin, VE-cadherin, and Ki67. Scale bar, 200 μm. Red lectin was injected via the tail vein, illustrating a direct connection of the HVP kidney patch with the host venous system. Scale bar, 50 μm. (E and F) A 6+-week-old HVP kidney patch was harvested for ex vivo optical mapping and observed under brightfield (E) to be electrically responsive to stimulation (F). (G) A unique developmental window for HVP engraftment occurred at day 6 of differentiation; day 3 mesodermal MESP1 + cells resulted in teratomas, and day 10 late NKX2.5 + heart precursors failed to form a ventricular muscle graft patch (n = 4). (H and I) Cross-sectional area of a 6+-week-old HVP kidney graft patch during relaxation (i.e., diastolic phase; H) and maximal contraction (i.e., systolic phase; I), respectively, as imaged by ultrasound. (J) Measured cross-sectional area of the graft patch during diastole and systole.

Article Snippet: Tissue sections were subsequently stained with various antibodies against αSMA (Thermo Scientific, 14-9760-82, USA); Brachyury (R&D Systems, AF2085, USA); CD31 (Cell Signaling Technology, mab 3528, USA); cTnI (Abcam, ab47003, UK); connexin 43 (Abcam, ab11370, UK); cTnT (Thermo Scientific, MS-295-P1, USA); GFP (Abcam, ab13970, UK); HCN4 (R&D Systems, MAB8138, USA); human fibronectin (R&D Systems, AF1918, USA); ISL1 (DSHB, 39.3F7, USA); Ki67 (BD Pharmingen, 550609, USA); human mitochondria (Millipore, mab1273, USA); laminin (DSHB, 2E8, USA); Mesp1 (Abcam, ab77013, UK); MLC2v (Proteintech, 10906-1-AP); MLC2a (Synaptic Systems, 311-011); MYH11 (Sigma, HPA015310, USA); and VE-cadherin (R&D Systems, USA).

Techniques: In Vivo, Transplantation Assay, Injection, Electron Microscopy, Immunofluorescence, Staining, Membrane, Ex Vivo

Journal: Molecular Therapy

Article Title: Human ISL1 + Ventricular Progenitors Self-Assemble into an In Vivo Functional Heart Patch and Preserve Cardiac Function Post Infarction

doi: 10.1016/j.ymthe.2018.02.012

Figure Lengend Snippet: In Vivo Transplantation of HVPs into Healthy and Injured Hearts (A) White dashed box denotes the location of the HVP graft 8 months following intramyocardial injection into a healthy heart. (B) Immunofluorescence staining showed that HVP ventricular grafts in the heart expressed NKX2.5-GFP (green), MLC2v, connexin 43, and MHY11; a minority of cells co-expressed MLC2a and was negative for HCN4 and ISL1. CD31 was only present outside the graft. (C) The white dashed box identifies the localization and the engraftment of the xeno tissue 2 months after injection of the HVPs into the border zone of MI hearts. Red asterisks denote infarction site. (D) Immunofluorescence staining showed that HVP ventricular grafts in post-MI hearts co-expressed NKX2.5-GFP (green), connexin 43, human nuclei, MLC2v, MHY11, human fibronectin, and laminin. A minority of cells was positive for ki67; MLC2a, ISL1, and HCN4 staining was not observed. Endothelial marker CD31 was detected in close proximity to the HVP graft. (E) High-resolution imaging of the border zone of post-MI HVP grafts demonstrated engrafted, differentiated cardiomyocytes of human origin, with well-organized sarcomeres (stains of cell membrane [WGA, green], human mitochondria [red], and F-actin [orange]). (F) MRI assessment of post-MI hearts 2 months following HVP transplantation showed improved global and local function in comparison with placebo. Representative short axis images show wall thickness at the site of induced infarction (arrows). LV, left ventricle; RV, right ventricle. (G) Mean measurements ± SD of wall thickening, ejection fraction, and volume during end diastole (EDV) and end systole (ESV). *p < 0.05; **p < 0.01; n/s, not significant.

Article Snippet: Tissue sections were subsequently stained with various antibodies against αSMA (Thermo Scientific, 14-9760-82, USA); Brachyury (R&D Systems, AF2085, USA); CD31 (Cell Signaling Technology, mab 3528, USA); cTnI (Abcam, ab47003, UK); connexin 43 (Abcam, ab11370, UK); cTnT (Thermo Scientific, MS-295-P1, USA); GFP (Abcam, ab13970, UK); HCN4 (R&D Systems, MAB8138, USA); human fibronectin (R&D Systems, AF1918, USA); ISL1 (DSHB, 39.3F7, USA); Ki67 (BD Pharmingen, 550609, USA); human mitochondria (Millipore, mab1273, USA); laminin (DSHB, 2E8, USA); Mesp1 (Abcam, ab77013, UK); MLC2v (Proteintech, 10906-1-AP); MLC2a (Synaptic Systems, 311-011); MYH11 (Sigma, HPA015310, USA); and VE-cadherin (R&D Systems, USA).

Techniques: In Vivo, Transplantation Assay, Injection, Immunofluorescence, Staining, Marker, Imaging, Membrane, Comparison

Journal: Investigative Ophthalmology & Visual Science

Article Title: Thy1 Associates with the Cation Channel Subunit HCN4 in Adult Rat Retina

doi: 10.1167/iovs.11-9307

Figure Lengend Snippet: Colocalization of Thy1 and HCN4 in ganglion cell layer somata. (A–C) Single optical section through the ganglion cell layer of a retina incubated in polyclonal anti-HCN4 primary and DyLight 488-conjugated secondary, anti-Thy1 primary, and DyLight 549-conjugated secondary, and Nissl stain. Fluorescence from DyLight 488 (A), DyLight 549 (B), and Nissl (C) assigned to the green, red, and blue color channels, respectively, and merged in (C). (D–F) Single optical section through the ganglion cell layer of a different portion of the same retina, processed as in (A–C) except that the anti-HCN4 primary was preincubated with immunogen. The scale bar in (C) (20 μm) applies to (C) alone; the scale bar in (F) (20 μm) applies to (A, B, D–F).

Article Snippet: The other anti-HCN4 antibody was clone N114/10 (73-150; UC Davis/NIH NeuroMab Facility, Davis, CA), a monoclonal mouse IgG1 raised against a fusion protein of GST and amino acids 1019 to 1108 of rat HCN4.

Techniques: Incubation, Staining, Fluorescence

Journal: Investigative Ophthalmology & Visual Science

Article Title: Thy1 Associates with the Cation Channel Subunit HCN4 in Adult Rat Retina

doi: 10.1167/iovs.11-9307

Figure Lengend Snippet: Colocalization of Thy1 and HCN4 in ganglion cell somata. (A–C) A single optical section through the ganglion cell layer of a retina incubated in monoclonal anti-HCN4 primary and DyLight 488-conjugated secondary (A, C), anti-Thy1 primary and DyLight 549-conjugated secondary (B, C), and anti-Brn3a primary and DyLight 649-conjugated secondary (C). Fluorescence from DyLight 488, 549, and 649 were assigned to the green, red, and blue color channels, respectively, and are merged in (C). (D–F) Single optical section through the ganglion cell layer of a different portion of the same retina, processed as in (A–C) except that the anti-Thy1 primary was not applied. The scale bar in (C) (20 μm) applies to (C) alone; the scale bar in (F) (20 μm) applies to (A, B, D–F).

Article Snippet: The other anti-HCN4 antibody was clone N114/10 (73-150; UC Davis/NIH NeuroMab Facility, Davis, CA), a monoclonal mouse IgG1 raised against a fusion protein of GST and amino acids 1019 to 1108 of rat HCN4.

Techniques: Incubation, Fluorescence

Journal: Investigative Ophthalmology & Visual Science

Article Title: Thy1 Associates with the Cation Channel Subunit HCN4 in Adult Rat Retina

doi: 10.1167/iovs.11-9307

Figure Lengend Snippet: Coimmunoprecipitation of Thy1 and HCN4 by beads coated with anti-Thy1 antibody. Protein G-coated beads (Dynabeads, 100.03D; Invitrogen) were BS3-cross-linked to Thy-1 antibody and then incubated with lysate. Proteins were eluted from these beads, analyzed with Western blot, and sequentially probed for Thy1 (lanes a–d), HCN4 (lanes e–h) using polyclonal antibody, and ABCR (lanes i–l). Before the second and third probes, the previously applied primary antibodies were stripped with glycine. The material aliquoted into each SDS-PAGE gel lane is specified underneath by the combination of + and −. Lanes a, e, and i are material eluted from raw beads (without antibody coating and without lysate). Lanes b, f, and j are material eluted from antibody-coated beads without lysate. Lanes c, g, and k are proteins eluted from Thy1 antibody-coated beads incubated with lysate. Lanes d, h, and l are lysate alone. The migration distances of the molecular weight standards along the left apply to the lanes probed by Thy1 antibody. Those along the right apply to the lanes probed by HCN4 and ABCR antibodies.

Article Snippet: The other anti-HCN4 antibody was clone N114/10 (73-150; UC Davis/NIH NeuroMab Facility, Davis, CA), a monoclonal mouse IgG1 raised against a fusion protein of GST and amino acids 1019 to 1108 of rat HCN4.

Techniques: Incubation, Western Blot, SDS Page, Migration, Molecular Weight

Journal: Investigative Ophthalmology & Visual Science

Article Title: Thy1 Associates with the Cation Channel Subunit HCN4 in Adult Rat Retina

doi: 10.1167/iovs.11-9307

Figure Lengend Snippet: Coimmunoprecipitation of Thy1 and HCN4 by incubation of beads, coated with monoclonal anti-HCN4 antibody, with lysates, formatted as in Figure 4. The proteins eluted from these beads were separated by SDS-PAGE, with samples of the lysate and molecular weight standards in adjacent lanes. The gels were analyzed with Western blot and the blots were separated into three segments by slicing at the level of the 185 kD standard and at approximately 50 kD. The upper, middle, and lower segments were probed with antibodies against ABCR, HCN4 (polyclonal), and Thy1, respectively. The ABCR and Thy1 antibodies were visualized with Fc fragment-specific, DyLight 649-conjugated, goat anti-mouse IgG. The HCN4 antibody was visualized with detection reagent (Clean-Blot IP; Thermo Scientific).

Article Snippet: The other anti-HCN4 antibody was clone N114/10 (73-150; UC Davis/NIH NeuroMab Facility, Davis, CA), a monoclonal mouse IgG1 raised against a fusion protein of GST and amino acids 1019 to 1108 of rat HCN4.

Techniques: Incubation, SDS Page, Molecular Weight, Western Blot

Journal: Cells

Article Title: Lactate-Induced HBEGF Shedding and EGFR Activation: Paving the Way to a New Anticancer Therapeutic Opportunity

doi: 10.3390/cells13181533

Figure Lengend Snippet: List of the antibodies used for protein detection.

Article Snippet: IF, primary , E-CAD , mouse , MAB8138 , R&D System (f) , 1:100 , 16 h (4 °C).

Techniques: