Journal: Acta Histochemica et Cytochemica

Article Title: Immunohistochemical Demonstration of Lymphatic Vessels in Adult Zebrafish

doi: 10.1267/ahc.12027

Figure Lengend Snippet: Fluorescence micrographs of tissue sections of zebrafish ovary ( a ) immunostained for Prox1 (red in a ) and plakoglobin (green in a ) and of murine ovary ( b ) immunostained for LYVE-1 (red in b ) and CD31 (green in b ). a : A lymphatic vessel (L) lined with endothelial cells, showing Prox1-immunopositive cellular nuclei and plakoglobin-immunopositive endothelial contours, embraces arterioles showing plakoglobin-immunopositive endothelium (arrow). b : The arterioles with CD31-immunopositive endothelium are seen to be surrounded by the LYVE-1-immunopositive lymphatic. The lymphatic shows a closed configuration presumably due to the high stromal tissue pressure. Bars=20 µm.

Article Snippet: The sections from fishes and murine ovaries were incubated in 10% normal donkey serum, and then in anti-prox1 antibody (Abcam), or in a mixture of an antibody to Prox1 or LYVE-1 (Angiobio, Del Mar, CA, USA), another lymphatic endothelial molecule [ , , ], together with an antibody to plakoglobin (PROGEN Biotechnik, Heidelberg, Germany) or CD31 (BD Biosciences Pharmingen, San Jose, CA, USA) at 4°C overnight.

Techniques: Fluorescence

Journal: eLife

Article Title: Lymphangiogenic therapy prevents cardiac dysfunction by ameliorating inflammation and hypertension

doi: 10.7554/eLife.58376

Figure Lengend Snippet: SNPs mapping to lymphatic endothelial markers associate with cardiac comorbidities. SNPs in human genetic loci, where lymphatic endothelial marker genes VEGFC , LYVE1 , FLT4 are located, are associated with cardiovascular and metabolic phenotypes. MRVI1 , murine retrovirus integration site one homolog. ADM , adrenomedullin. CTR9 , RNA polymerase-associated protein CTR9 homolog. MGAT1 , alpha-1,3-mannosyl-glycoprotein 2-beta-N-acetylglucosaminyltransferase. SCGB3A1 , Secretoglobin Family 3A Member 1. OR2Y1 , olfactory receptor family two subfamily Y member 1.

Article Snippet: The RNA was either directly used for qPCR with TaqMan RNA to Ct 1-step kit (ThermoFisher) or cDNA was synthesized from the RNA using high capacity cDNA reverse transcription kit (ThermoFisher) and used for qPCR. qPCR was conducted using TaqMan probes ( Nppb , Mm01255770_g1; Pdpn , Mm01348912_g1; Lyve1 , Mm00475056_m1; Flt4 , Mm01292604_m1; Vegfc , Mm00437310_m1; Col1a1 , Mm00801666; Col1a2 , Mm00483888; Col3a1 , Mm00802300_m1; Fn1 , Mm01256744; Cd68 , Mm03047343_m1; Cxcl10 , Mm00445235_m1; Ptprc , Mm01293577_m1; Itgax , Mm00498701_m1; Itgam , Mm00434455_m1; Il1b , Mm00434228_m1; Il6 , Mm00446190_m1; Ccl6 , Mm01302419_m1; Ccl4 , Mm00443111_m1; Ccl3 , Mm00441259_g1; Ace2 , Mm01159006_m1; Hprt , Mm03024075_m1), TaqMan gene expression master mix (ThermoFisher) and QuantStudio 7 Flex Real-Time PCR System.

Techniques: Marker, Clinical Proteomics, Concentration Assay

Journal: eLife

Article Title: Lymphangiogenic therapy prevents cardiac dysfunction by ameliorating inflammation and hypertension

doi: 10.7554/eLife.58376

Figure Lengend Snippet: ( A–D ) Expression of lymphatic endothelial marker genes VEGFC ( A ), LYVE1 ( B ), PDPN ( C ), FLT4 ( D ) in hearts from healthy human donors (HH) and hearts from patients with chronic heart failure (CHF). The data were normalized to the expression of the housekeeping gene HPRT1 and represented as fold change from the HH group ( n = 16–18 for HH and n = 10–11 for CHF). Data are mean ± s.d. Student’s t -test was used for statistics. * p <0.05. Figure 1—source data 1. Clinical information. Figure 1—source data 2. Raw data supporting .

Article Snippet: The RNA was either directly used for qPCR with TaqMan RNA to Ct 1-step kit (ThermoFisher) or cDNA was synthesized from the RNA using high capacity cDNA reverse transcription kit (ThermoFisher) and used for qPCR. qPCR was conducted using TaqMan probes ( Nppb , Mm01255770_g1; Pdpn , Mm01348912_g1; Lyve1 , Mm00475056_m1; Flt4 , Mm01292604_m1; Vegfc , Mm00437310_m1; Col1a1 , Mm00801666; Col1a2 , Mm00483888; Col3a1 , Mm00802300_m1; Fn1 , Mm01256744; Cd68 , Mm03047343_m1; Cxcl10 , Mm00445235_m1; Ptprc , Mm01293577_m1; Itgax , Mm00498701_m1; Itgam , Mm00434455_m1; Il1b , Mm00434228_m1; Il6 , Mm00446190_m1; Ccl6 , Mm01302419_m1; Ccl4 , Mm00443111_m1; Ccl3 , Mm00441259_g1; Ace2 , Mm01159006_m1; Hprt , Mm03024075_m1), TaqMan gene expression master mix (ThermoFisher) and QuantStudio 7 Flex Real-Time PCR System.

Techniques: Expressing, Marker

Journal: eLife

Article Title: Lymphangiogenic therapy prevents cardiac dysfunction by ameliorating inflammation and hypertension

doi: 10.7554/eLife.58376

Figure Lengend Snippet: ( A ) Pharmacokinetic analysis of VEGFC C156S in mice ( n = 2 per dose group). ( B ) Plasma VEGFC concentration measured by ELISA at euthanasia ( n = 10–17/group). ( C–S ) Mice were infused with saline + BSA, angiotensin II + BSA, or angiotensin II + VEGFC C156S as described in . ( C ) Representative M-mode echocardiography images for each group. ( D–G ) Echocardiography parameters cardiac output ( D ), stroke volume ( E ), Left Ventricular Posterior Wall thickness at diastole (LVPWd) ( F ) and heart rate ( G ) are shown ( n = 15–24/group). ( H–J ) Hearts were arrested in diastole, fixed, sectioned, and stained with wheat germ agglutinin (WGA). ( H ) Representative images of WGA stain for each group. Scale bar, 100 μm. ( I ) Cardiomyocyte size was assessed ( n = 7–9/group). ( J ) Analysis of cell size variance coefficient from WGA-stained mouse heart sections ( n = 21–27/group; a linear mixed model was used for statistics). ( K–N ) Protein lysates were prepared from mouse hearts and immunoblotted for podoplanin, p-Akt, Akt, p-Erk1/2, Erk1/2, and GAPDH. ( K ) Representative immunoblots. ( L–N ) Densitometric quantification of podoplanin ( L ), p-Akt to Akt ratio ( M ) and p-Erk to Erk ratio ( N ) ( n = 7–9/group). ( O ) Representative images of the whole mount stain of lyve1-positive lymphatic vessels in ear skin for each group. Scale bar, 100 μm. ( P ) Quantification of lyve1-positive lymphatic vessel diameter in ear skin ( n = 737–880 vessel/group; a linear mixed model was used for statistics). ( Q ) Ear skin samples were fixed, sectioned, and stained for lyve1(green) and DAPI (blue). Representative images of lyve1-positive lymphatic vessels in ear skin cross-section for each group. Scale bar, 200 μm. ( R ) Quantification of lyve1-positive lymphatic vessel density (normalized to total area) in ear skin ( n = 6–8 animals/group, n = 6–8 sections/animal; a linear mixed model was used for statistics). Ctrl, control. AngII, angiotensin II. VEGFC, VEGFCc156s. Data are mean ± s.d. One-way ANOVA with Bonferroni posthoc was used for statistics for all figures except 2J, 2P and 2R (a linear mixed model was used for statistics). * p <0.05, ** p <0.01, *** p <0.001, **** p <0.0001, n.s. not significant.

Article Snippet: The RNA was either directly used for qPCR with TaqMan RNA to Ct 1-step kit (ThermoFisher) or cDNA was synthesized from the RNA using high capacity cDNA reverse transcription kit (ThermoFisher) and used for qPCR. qPCR was conducted using TaqMan probes ( Nppb , Mm01255770_g1; Pdpn , Mm01348912_g1; Lyve1 , Mm00475056_m1; Flt4 , Mm01292604_m1; Vegfc , Mm00437310_m1; Col1a1 , Mm00801666; Col1a2 , Mm00483888; Col3a1 , Mm00802300_m1; Fn1 , Mm01256744; Cd68 , Mm03047343_m1; Cxcl10 , Mm00445235_m1; Ptprc , Mm01293577_m1; Itgax , Mm00498701_m1; Itgam , Mm00434455_m1; Il1b , Mm00434228_m1; Il6 , Mm00446190_m1; Ccl6 , Mm01302419_m1; Ccl4 , Mm00443111_m1; Ccl3 , Mm00441259_g1; Ace2 , Mm01159006_m1; Hprt , Mm03024075_m1), TaqMan gene expression master mix (ThermoFisher) and QuantStudio 7 Flex Real-Time PCR System.

Techniques: Clinical Proteomics, Concentration Assay, Enzyme-linked Immunosorbent Assay, Saline, Staining, Western Blot, Control

Journal: eLife

Article Title: Lymphangiogenic therapy prevents cardiac dysfunction by ameliorating inflammation and hypertension

doi: 10.7554/eLife.58376

Figure Lengend Snippet: ( A ) Design of Prox1-eGFP KI mouse model. The model was generated using CRISPR/Cas9 Technology, and an IRES-eGFP cassette was inserted into the 3’UTR of the endogenous Prox1 locus. ( B ) Representative images of lymphatic vessels with eGFP fluorescence in the diaphragm(top) and heart(bottom) from Prox1-eGFP mice. Scale bar, 100 μm. ( C ) Representative images of lymphatic vessels in the heart (left) of Prox1-eGFP mice stained with GFP (green), Lyve1 (lymphatic endothelial marker, red) and DAPI(blue), and lymphatic vessels in the diaphragm(right) of prox1-eGFP mice stained with GFP (green) and Lyve1 (red). The GFP stain overlapped with Lyve1 stain. Scale bar, 50 μm. ( D ) Schematic illustration of cardiomyocytes, heart lymphatic endothelial cell (heart LEC), heart blood endothelial cell (heart BEC) and diaphragm lymphatic endothelial cell (diaphragm LEC) isolation from Prox1-eGFP mice and representative flow cytometry plots. ( E ) Principal component analysis (PCA) for RNA-seq data, with tissue and treatment conditions denoted by color and shape, respectively. ( F–G ) Heat map visualization of all FDR-significant differential expression changes in genes comparing angII and ctrl groups (angII vs ctrl), angII+VEGFC and angII groups (angII+VEGFC vs angII), and angII+VEGFC and ctrl groups (angII+VEGFC vs ctrl) in heart BECs and cardiomyocytes. The differential expression test-statistics are indicated by the shading in the heatmap. ( H ) Summary of signaling pathway analysis in angII vs ctrl and angII+VEGFC vs angII comparisons in different tissue and cell types. The unit for x-axis (-log10(‘weight p-value’)) indicates significance level and does not indicate directionality of the change. ( I ) Causal reasoning analysis indicated that angiotensin II is a causal factor for the transcriptional changes in cardiomyocytes in both angII vs ctrl comparison and angII+VEGFC vs ctrl comparison. FDR less than 0.05 was defined as significant. ( J ) Top five common FDR-significant causal proteins for heart LECs and the common FDR-significant causal protein for diaphragm LECs in angII vs ctrl comparison and angII+VEGFC vs angII comparison from causal reasoning analysis. The directionality of the effect was shown in the table as activation (+) and inactivation (-). ( K ) KC/GRO levels in native mouse skin lysates. The values of KC/GRO were log-transformed, and the values were normalized to the ctrl group and represented as fold change ( n = 7–9/group). Ctrl, control. AngII, angiotensin II. VEGFC, VEGFCc156s. Data are mean ± s.d. One-way ANOVA with Tukey’s posthoc was used for log-transformed data. n.s. not significant.

Article Snippet: The RNA was either directly used for qPCR with TaqMan RNA to Ct 1-step kit (ThermoFisher) or cDNA was synthesized from the RNA using high capacity cDNA reverse transcription kit (ThermoFisher) and used for qPCR. qPCR was conducted using TaqMan probes ( Nppb , Mm01255770_g1; Pdpn , Mm01348912_g1; Lyve1 , Mm00475056_m1; Flt4 , Mm01292604_m1; Vegfc , Mm00437310_m1; Col1a1 , Mm00801666; Col1a2 , Mm00483888; Col3a1 , Mm00802300_m1; Fn1 , Mm01256744; Cd68 , Mm03047343_m1; Cxcl10 , Mm00445235_m1; Ptprc , Mm01293577_m1; Itgax , Mm00498701_m1; Itgam , Mm00434455_m1; Il1b , Mm00434228_m1; Il6 , Mm00446190_m1; Ccl6 , Mm01302419_m1; Ccl4 , Mm00443111_m1; Ccl3 , Mm00441259_g1; Ace2 , Mm01159006_m1; Hprt , Mm03024075_m1), TaqMan gene expression master mix (ThermoFisher) and QuantStudio 7 Flex Real-Time PCR System.

Techniques: Generated, CRISPR, Fluorescence, Staining, Marker, Isolation, Flow Cytometry, RNA Sequencing, Quantitative Proteomics, Comparison, Activation Assay, Transformation Assay, Control

Journal: eLife

Article Title: Lymphangiogenic therapy prevents cardiac dysfunction by ameliorating inflammation and hypertension

doi: 10.7554/eLife.58376

Figure Lengend Snippet: ( A ) Kidneys from the mice described in were fixed, sectioned, and stained. Representative H and E stains and Picrosirius red (PSR) stains of mouse kidney sections are shown ( n = 8–10/group). Black asterisk indicates vascular hypertrophy, green asterisk indicates interstitial mononuclear cell infiltrates and red asterisk indicates tubular basophilia. Scale bar, 100 μm. ( B ) A summary of pathological grading of kidney damage based on H and E stained kidney sections. ( C–D ) Quantification of PSR-positive area ( C ) and perivascular PSR-positive area ( D ) (normalized to total area) on PSR-stained kidney sections ( n = 8–10/group). ( E–F ) RNA was extracted from mouse kidneys and qPCR was performed for Vegfc ( E ), Flt4 ( F ) and normalized to the expression of housekeeping gene Hprt in mouse kidneys. The data were normalized to the ctrl group and represented as fold change ( n = 16–21/group). ( G ) Mouse kidneys were fixed, OCT embedded, sectioned, and stained for lyve1 (green), smooth muscle actin (red), and DAPI (light blue). Representative images for each group are shown. Scale bar, 100 μm. ( H ) Quantification of lyve1-positive lymphatic vessel density (normalized to total area) in stained mouse kidney sections ( n = 7–10/group). Ctrl, control. AngII, angiotensin II. VEGFC, VEGFCc156s. Data are mean ± s.d. One-way ANOVA with Bonferroni posthoc was used for statistics. * p <0.05, ** p <0.01, *** p <0.001, **** p <0.0001, n.s. not significant.

Article Snippet: The RNA was either directly used for qPCR with TaqMan RNA to Ct 1-step kit (ThermoFisher) or cDNA was synthesized from the RNA using high capacity cDNA reverse transcription kit (ThermoFisher) and used for qPCR. qPCR was conducted using TaqMan probes ( Nppb , Mm01255770_g1; Pdpn , Mm01348912_g1; Lyve1 , Mm00475056_m1; Flt4 , Mm01292604_m1; Vegfc , Mm00437310_m1; Col1a1 , Mm00801666; Col1a2 , Mm00483888; Col3a1 , Mm00802300_m1; Fn1 , Mm01256744; Cd68 , Mm03047343_m1; Cxcl10 , Mm00445235_m1; Ptprc , Mm01293577_m1; Itgax , Mm00498701_m1; Itgam , Mm00434455_m1; Il1b , Mm00434228_m1; Il6 , Mm00446190_m1; Ccl6 , Mm01302419_m1; Ccl4 , Mm00443111_m1; Ccl3 , Mm00441259_g1; Ace2 , Mm01159006_m1; Hprt , Mm03024075_m1), TaqMan gene expression master mix (ThermoFisher) and QuantStudio 7 Flex Real-Time PCR System.

Techniques: Staining, Expressing, Control

Journal: eLife

Article Title: Lymphangiogenic therapy prevents cardiac dysfunction by ameliorating inflammation and hypertension

doi: 10.7554/eLife.58376

Figure Lengend Snippet: ( A–D ) Urine was collected 4.5 weeks after minipump implantation. Urine volume ( A ), Urine non-esterified fatty acid (NEFA) levels ( B ), total urinary protein ( C ) and albumin to creatinine ratio (ACR) ( D ) are shown ( n = 7–10/group). ( E–F ) RNA was extracted from whole kidney, and quantitative RT-PCR was performed for Lyve1 ( E ) and Pdpn ( F ) and normalized to the expression of housekeeping gene Hprt . The data were normalized to the control group and represented as fold change ( n = 16–21/group). Ctrl, control. AngII, angiotensin II. VEGFC, VEGFCc156s. Data are mean ± s.d. One-way ANOVA with Bonferroni posthoc was used for statistics. * p <0.05, ** p <0.01, **** p <0.0001, n.s. not significant. Figure 6—source data 1. Raw data supporting .

Article Snippet: The RNA was either directly used for qPCR with TaqMan RNA to Ct 1-step kit (ThermoFisher) or cDNA was synthesized from the RNA using high capacity cDNA reverse transcription kit (ThermoFisher) and used for qPCR. qPCR was conducted using TaqMan probes ( Nppb , Mm01255770_g1; Pdpn , Mm01348912_g1; Lyve1 , Mm00475056_m1; Flt4 , Mm01292604_m1; Vegfc , Mm00437310_m1; Col1a1 , Mm00801666; Col1a2 , Mm00483888; Col3a1 , Mm00802300_m1; Fn1 , Mm01256744; Cd68 , Mm03047343_m1; Cxcl10 , Mm00445235_m1; Ptprc , Mm01293577_m1; Itgax , Mm00498701_m1; Itgam , Mm00434455_m1; Il1b , Mm00434228_m1; Il6 , Mm00446190_m1; Ccl6 , Mm01302419_m1; Ccl4 , Mm00443111_m1; Ccl3 , Mm00441259_g1; Ace2 , Mm01159006_m1; Hprt , Mm03024075_m1), TaqMan gene expression master mix (ThermoFisher) and QuantStudio 7 Flex Real-Time PCR System.

Techniques: Quantitative RT-PCR, Expressing, Control

Journal: eLife

Article Title: Lymphangiogenic therapy prevents cardiac dysfunction by ameliorating inflammation and hypertension

doi: 10.7554/eLife.58376

Figure Lengend Snippet: Mice were infused with saline + BSA, saline+ VEGFC C156S , angiotensin II + BSA, or angiotensin II + VEGFC C156S as described in . ( A ) Plasma VEGFC concentration measured by ELISA at euthanasia (1 week post-treatment) ( n = 12–17/group). ( B–C ) Plasma hemoglobin concentration ( B ) and hematocrit percentage ( C ) measured at euthanasia (1 week post-treatment) ( n = 13–18/group). ( D ) RNA was extracted from mouse kidneys and qPCR was performed for Ace2 and normalized to Hprt . The data were normalized to the control (ctrl) group and represented as fold change ( n = 12–18/group). ( E ) Urine was collected at day 5 after minipump implantation. Urine albumin to creatinine ratio (ACR) was measured and is shown ( n = 10–17/group). ( F ) RNA was extracted from whole kidney, and quantitative RT-PCR was performed for Lyve1 and normalized to the expression of housekeeping gene Hprt . The data were normalized to the ctrl group and represented as fold change ( n = 12–18/group). ( G ) Mouse kidneys were fixed, OCT embedded, sectioned and stained for lyve1 (green), smooth muscle actin (red) and DAPI (blue). Representative images for each group are shown. Scale bar, 200 μm. ( H ) Quantification of lyve1-positive lymphatic vessel density (normalized to total area) in stained mouse kidney sections ( n = 11–20/group). ( I ) Ear skin samples were fixed, sectioned, and stained for lyve1(green) and DAPI (blue). Representative images of lyve1-positive lymphatic vessels in ear skin cross-section for each group. Scale bar, 200 μm. ( J ) Quantification of lyve1-positive lymphatic vessel density (normalized to total area) in ear skin ( n = 6–10 animals/group, n = 5–7 sections/animal; a linear mixed model was used for statistics). ( K ) Mouse hearts were fixed, OCT embedded, sectioned and stained, for podoplanin, smooth muscle actin, and DAPI (light blue). Representative images of podoplanin-positive lymphatic vessels (green) and smooth muscle actin-positive arteries (red) for each group were shown. Scale bar, 200 μm. ( L ) Quantification of podoplanin-positive lymphatic vessel density (normalized to total area and represented as fold change to ctrl group) in mouse hearts ( n = 7–10 animal/group, n = 4–6 sections/animal; a linear mixed model was used for statistics). ( M ) Representative M-mode echocardiography images for each group. ( N–O ) Echocardiography parameters heart rate ( N ) and Left Ventricular Posterior Wall thickness at diastole (LVPWd) ( O ) are shown ( n = 13–17/group). ( P–Q ) RNA was isolated from the mouse hearts, and quantitative RT-PCR was performed for Col1a2 ( P ) and Fn1 ( Q ) and normalized to the expression of housekeeping gene Hprt . The data were normalized to ctrl group and represented as fold change ( n = 6–8/group). AngII, angiotensin II. VEGFC, VEGFCc156s. Data are mean ± s.d. One-way ANOVA with Bonferroni posthoc was used for all figures except 7J and 7L (a linear mixed model was used for statistics). * p <0.05, ** p <0.01, *** p <0.001, **** p <0.0001, n.s. not significant.

Article Snippet: The RNA was either directly used for qPCR with TaqMan RNA to Ct 1-step kit (ThermoFisher) or cDNA was synthesized from the RNA using high capacity cDNA reverse transcription kit (ThermoFisher) and used for qPCR. qPCR was conducted using TaqMan probes ( Nppb , Mm01255770_g1; Pdpn , Mm01348912_g1; Lyve1 , Mm00475056_m1; Flt4 , Mm01292604_m1; Vegfc , Mm00437310_m1; Col1a1 , Mm00801666; Col1a2 , Mm00483888; Col3a1 , Mm00802300_m1; Fn1 , Mm01256744; Cd68 , Mm03047343_m1; Cxcl10 , Mm00445235_m1; Ptprc , Mm01293577_m1; Itgax , Mm00498701_m1; Itgam , Mm00434455_m1; Il1b , Mm00434228_m1; Il6 , Mm00446190_m1; Ccl6 , Mm01302419_m1; Ccl4 , Mm00443111_m1; Ccl3 , Mm00441259_g1; Ace2 , Mm01159006_m1; Hprt , Mm03024075_m1), TaqMan gene expression master mix (ThermoFisher) and QuantStudio 7 Flex Real-Time PCR System.

Techniques: Saline, Clinical Proteomics, Concentration Assay, Enzyme-linked Immunosorbent Assay, Control, Quantitative RT-PCR, Expressing, Staining, Isolation

Journal: eLife

Article Title: Lymphangiogenic therapy prevents cardiac dysfunction by ameliorating inflammation and hypertension

doi: 10.7554/eLife.58376

Figure Lengend Snippet:

Article Snippet: The RNA was either directly used for qPCR with TaqMan RNA to Ct 1-step kit (ThermoFisher) or cDNA was synthesized from the RNA using high capacity cDNA reverse transcription kit (ThermoFisher) and used for qPCR. qPCR was conducted using TaqMan probes ( Nppb , Mm01255770_g1; Pdpn , Mm01348912_g1; Lyve1 , Mm00475056_m1; Flt4 , Mm01292604_m1; Vegfc , Mm00437310_m1; Col1a1 , Mm00801666; Col1a2 , Mm00483888; Col3a1 , Mm00802300_m1; Fn1 , Mm01256744; Cd68 , Mm03047343_m1; Cxcl10 , Mm00445235_m1; Ptprc , Mm01293577_m1; Itgax , Mm00498701_m1; Itgam , Mm00434455_m1; Il1b , Mm00434228_m1; Il6 , Mm00446190_m1; Ccl6 , Mm01302419_m1; Ccl4 , Mm00443111_m1; Ccl3 , Mm00441259_g1; Ace2 , Mm01159006_m1; Hprt , Mm03024075_m1), TaqMan gene expression master mix (ThermoFisher) and QuantStudio 7 Flex Real-Time PCR System.

Techniques: Recombinant, Sequencing, Enzyme-linked Immunosorbent Assay, Reverse Transcription, Gene Expression, Real-time Polymerase Chain Reaction, Western Blot, Plasmid Preparation, Software, Microinjection