Journal: Developmental Cell

Article Title: Lineage-tracing hematopoietic stem cell origins in vivo to efficiently make human HLF+ HOXA+ hematopoietic progenitors from pluripotent stem cells

doi: 10.1016/j.devcel.2024.03.003

Figure Lengend Snippet: Genetic lineage tracing reveals that artery endothelial cells generate HSCs in vivo (A) Experimental strategy. DA, dorsal aorta; FL, fetal liver; PB, peripheral blood; BM, bone marrow; E, embryonic day; P, postnatal day. (B) Mass spectrometry quantification of ( Z )-4OHT levels in plasma of female adult Cx40-CreERT2 mice that intraperitoneally injected with ( Z )-4OHT. (C) Cx40 and CreERT2 in situ staining of E8.5 Cx40-CreERT2 mouse embryos, using hybridization chain reaction v3.0 (HCR3). Arrows: paired dorsal aortae. Ant, anterior; post, posterior. (D) scRNA-seq of the entire E8.5 mouse embryo. (E–J) Arteries were lineage-traced in Cx40-CreERT2 ; Ai6 ( ZsGreen reporter) embryos by administering 4OHT at E8.5. The Cx40-CreERT2 allele also encodes RFP , which was used to visualize Cx40+ cells. (E, G, and I) Immunostaining and (F, H, and J) flow cytometry of E11.5 dorsal aorta, E11.5 yolk sac, and E16.5 fetal liver was performed. (K) Arteries were lineage-traced in Cx40-CreERT2 ; Ai6 ( ZsGreen reporter) embryos by administering a single 4OHT dose at the indicated times (E7.5–E12.5). Flow cytometry was performed to quantify artery-derived (i.e., ZsGreen+) HSCs in the E14.5–E18.5 fetal liver. Each dot: independent litter. For each time point, ≥8 independent embryos were analyzed. Inset: fetal liver HSCs labeled after E9.0 4OHT administration. (L) Arteries were lineage-traced in Efnb2-CreERT2 ; Ai6 ( ZsGreen reporter) embryos by administering 4OHT at E8.5. Flow cytometry was performed to quantify ZsGreen+ E14.5–E18.5 fetal liver HSCs. (M) Veins and capillaries were lineage-traced in Apj-CreERT2 ; Ai6 ( ZsGreen reporter) embryos by administering 4OHT at E9.5. Flow cytometry was performed to quantify ZsGreen+ E14.5–E18.5 fetal liver HSCs. Histograms depict the mean ± standard error of the mean (SEM). ∗ p < 0.05, ∗∗ p < 0.01. Scale bars, 50 μm. Related to Figures S1 and and Table S1 .

Article Snippet: HCR3 probe for mouse Gja5 ( Cx40 ), compatible with amplifier B3 , Molecular Instruments , Custom probe against Gja5 (sequence deposited at NCBI accession NM_001271628).

Techniques: In Vivo, Mass Spectrometry, Clinical Proteomics, Injection, In Situ, Staining, Hybridization, Immunostaining, Flow Cytometry, Derivative Assay, Labeling

Journal: Developmental Cell

Article Title: Lineage-tracing hematopoietic stem cell origins in vivo to efficiently make human HLF+ HOXA+ hematopoietic progenitors from pluripotent stem cells

doi: 10.1016/j.devcel.2024.03.003

Figure Lengend Snippet: Artery-derived HSCs are functional in vivo (A–E) Arteries were lineage-traced in Cx40-CreERT2 ; Ai6 ( ZsGreen reporter) embryos by administering 4OHT at either E8.0, E8.5, or E9.0. After embryos developed into adults, flow cytometry was performed to quantify ZsGreen+ cells in (B) and (C) peripheral blood and (C) and (D) bone marrow HSCs in 1- to 22-month-old adult mice. Line graphs depict the mean ± SEM. Related to Figure S3 .

Article Snippet: HCR3 probe for mouse Gja5 ( Cx40 ), compatible with amplifier B3 , Molecular Instruments , Custom probe against Gja5 (sequence deposited at NCBI accession NM_001271628).

Techniques: Derivative Assay, Functional Assay, In Vivo, Flow Cytometry

Journal: Developmental Cell

Article Title: Lineage-tracing hematopoietic stem cell origins in vivo to efficiently make human HLF+ HOXA+ hematopoietic progenitors from pluripotent stem cells

doi: 10.1016/j.devcel.2024.03.003

Figure Lengend Snippet: Artery-derived HSCs are functional in vivo upon transplantation (A and B) Arteries were lineage-traced by administering 4OHT to E8.5 Cx40-CreERT2 ; Ai6 ( ZsGreen reporter) embryos. B6, C57BL/6 mouse. (C and D) ZsGreen+ E16.5 fetal liver HSCs were (B) analyzed by flow cytometry and (C and D) transplanted into lethally irradiated primary recipient mice. 1–4 months post transplantation, flow cytometry was performed to quantify ZsGreen+ (C) peripheral blood cells and (D) bone marrow HSCs in primary recipients. (E and F) Bone marrow from primary recipient mice was transplanted into lethally irradiated secondary recipient mice. 1–4 months post transplantation, flow cytometry was performed to quantify ZsGreen+ (E) peripheral blood and (F) bone marrow HSCs in secondary recipients. Data depict the mean ± SEM. Each dot represents a single mouse. Related to Figure S4 .

Article Snippet: HCR3 probe for mouse Gja5 ( Cx40 ), compatible with amplifier B3 , Molecular Instruments , Custom probe against Gja5 (sequence deposited at NCBI accession NM_001271628).

Techniques: Derivative Assay, Functional Assay, In Vivo, Transplantation Assay, Flow Cytometry, Irradiation

Journal: Cell Communication and Signaling : CCS

Article Title: NO, via its target Cx37, modulates calcium signal propagation selectively at myoendothelial gap junctions

doi: 10.1186/1478-811X-12-33

Figure Lengend Snippet: Distribution of Cx37 within the internal elastic lamina. A . Representative 2-photon-image of a small resistance artery for visualization of the internal elastic lamina and the connexins. Cx37 (red) located in the small holes (dark dots) within the internal elastic lamina (autofluorescence, green). Arrows indicate some of the holes in which Cx37 could be detected; scale bar: 25 μm. B depicts the summary of n = 7-10 experiments (3 vessels each) revealing the percentage of holes in the internal elastic lamina that contain the different vascular Cx (*: p < 0.05 vs. Cx40; #: p < 0.05 vs. Cx45, NG).

Article Snippet: After washing twice, and perfusing the for 5 min with PBS the vessels were incubated and perfused for 2 hours with PBS containing 1% BSA and 0.3% Triton-X100, washed and perfused again with PBS-1% BSA and incubated overnight with the antibodies against Cx37 (10 μg/ml Alpha Diagnostics, Cat. No. Cx37A11-A), Cx43 (see above), Cx40 (3.3 μg/ml polyclonal rabbit anti mouse-Cx40, Alpha-Diagnostics, Cat. No. Cx40-A) or Cx45 (2.5 μg/ml polyclonal rabbit anti Cx45 c-Term, Zymed, Cat. No. 40–7000) and against CD31 (2.5 μg/ml monoclonal rat anti mouse-CD31, Abcam, Cat. No 56299; antibodies were applied from the endothelial and smooth muscle side).

Techniques:

Journal: Cell Communication and Signaling : CCS

Article Title: NO, via its target Cx37, modulates calcium signal propagation selectively at myoendothelial gap junctions

doi: 10.1186/1478-811X-12-33

Figure Lengend Snippet: Location of vascular Cx across the vessel wall. A . Confocal images of triple (Cx, α-actin, CD31) immunohistochemical stainings of Cx40, Cx45, Cx37, and Cx43 in small resistance arteries. Left panel: Overlay of a z-series in xy-direction, right panel: Cross section (slice) of the z-stack in yz-direction along the yellow lines in the z-stack. The arrows depict the Cx expression in ECL (yellow) or beyond EC and within SMC (SMCL, white), scale bars: 10 μm. B . Summary of the Cx distribution within the ECL and SMCL for all Cx (n = 4-8, at least 3 vessels each; *: p < 0.001).

Article Snippet: After washing twice, and perfusing the for 5 min with PBS the vessels were incubated and perfused for 2 hours with PBS containing 1% BSA and 0.3% Triton-X100, washed and perfused again with PBS-1% BSA and incubated overnight with the antibodies against Cx37 (10 μg/ml Alpha Diagnostics, Cat. No. Cx37A11-A), Cx43 (see above), Cx40 (3.3 μg/ml polyclonal rabbit anti mouse-Cx40, Alpha-Diagnostics, Cat. No. Cx40-A) or Cx45 (2.5 μg/ml polyclonal rabbit anti Cx45 c-Term, Zymed, Cat. No. 40–7000) and against CD31 (2.5 μg/ml monoclonal rat anti mouse-CD31, Abcam, Cat. No 56299; antibodies were applied from the endothelial and smooth muscle side).

Techniques: Immunohistochemical staining, Expressing

Journal: Cells

Article Title: Novel Blood Vascular Endothelial Subtype-Specific Markers in Human Skin Unearthed by Single-Cell Transcriptomic Profiling

doi: 10.3390/cells11071111

Figure Lengend Snippet: Molecular characterization of arteriole and post-arterial capillary endothelial cells. Exclusive expression of the arterial marker GJA5 by BVs (white arrowheads ( A )) and its overlay with HEY1 ( B ). The expression of ASS1 ( C ) and S100A4 ( D ) was restricted to the A and PAC clusters (white arrowheads). ( E ) Differential localization of ACKR1 and ASS1 discerns arterioles and post-arterial capillaries. Representative confocal images of section (upper) and whole-mount staining (lower) of ERG (green), ASS1 (red) and ACKR1 (white). Scale bars: 100 µm.

Article Snippet: After fixation in 4% paraformaldehyde (PFA), skin sections were incubated in blocking solution (5% donkey serum, 0.2% BSA, 0.3% Triton X-100 and 0.05% NaN 3 in PBS) for 1 h. Subsequently, the sections were incubated at 4 °C overnight with the respective primary antibodies, which include rabbit anti-human LYVE-1 (1:100, 102PA50AG, ReliaTech, Wolfenbüttel, Germany), biotinylated goat anti-human LYVE-1 (1:100, BAF2089, R&D, Minneapolis, MN, USA), mouse anti-human CD31 (1:100, clone JC70A, M0823, Dako, Santa Clara, CA, USA), rabbit anti-human CD31 (1:40, GTX110602, GeneTex, Irvine, CA, USA), rabbit anti-human ERG (1:100, ab92513, Abcam, Cambridge, UK), goat anti-human ERG (1:50, NB100-2472, Novus, Centennial, CO, USA), goat anti-human ACKR1 (1:200, NB100-2421, Novus), mouse anti-human ICAM1 (1:100, clone W-CAM-1, MA5-13021, Invitrogen, Waltham, MA, USA), sheep anti-human ICAM1 (1:100, AF720, R&D), rabbit anti-human HEY1 (1:100, ab22614, Abcam), mouse anti-human SELP (1:300, clone AK-6, ab6632, Abcam), goat anti-human EFNB2 (1:100, AF496, R&D), rabbit anti-human EGR2 (1:200, ab43020, Abcam), rabbit anti-human LRG1 (1:200, HPA001888, Atlas Antibodies, Bromma, Sweden), mouse anti-human GJA5 (1:250, clone 2F9A11, 37-8900, Invitrogen), mouse anti-human ASS1 (1:100, clone 2B10, ab124465, Abcam), rabbit anti-human S100A4 (1:200, ab41532, Abcam), goat anti-human SOX17 (1:10, AF1924, R&D) and mouse anti-human PLAUR (1:100, clone 62022, MAB807, R&D).

Techniques: Expressing, Marker, Staining

Journal: Cells

Article Title: Novel Blood Vascular Endothelial Subtype-Specific Markers in Human Skin Unearthed by Single-Cell Transcriptomic Profiling

doi: 10.3390/cells11071111

Figure Lengend Snippet: Summary of marker expression in different BEC subtypes.

Article Snippet: After fixation in 4% paraformaldehyde (PFA), skin sections were incubated in blocking solution (5% donkey serum, 0.2% BSA, 0.3% Triton X-100 and 0.05% NaN 3 in PBS) for 1 h. Subsequently, the sections were incubated at 4 °C overnight with the respective primary antibodies, which include rabbit anti-human LYVE-1 (1:100, 102PA50AG, ReliaTech, Wolfenbüttel, Germany), biotinylated goat anti-human LYVE-1 (1:100, BAF2089, R&D, Minneapolis, MN, USA), mouse anti-human CD31 (1:100, clone JC70A, M0823, Dako, Santa Clara, CA, USA), rabbit anti-human CD31 (1:40, GTX110602, GeneTex, Irvine, CA, USA), rabbit anti-human ERG (1:100, ab92513, Abcam, Cambridge, UK), goat anti-human ERG (1:50, NB100-2472, Novus, Centennial, CO, USA), goat anti-human ACKR1 (1:200, NB100-2421, Novus), mouse anti-human ICAM1 (1:100, clone W-CAM-1, MA5-13021, Invitrogen, Waltham, MA, USA), sheep anti-human ICAM1 (1:100, AF720, R&D), rabbit anti-human HEY1 (1:100, ab22614, Abcam), mouse anti-human SELP (1:300, clone AK-6, ab6632, Abcam), goat anti-human EFNB2 (1:100, AF496, R&D), rabbit anti-human EGR2 (1:200, ab43020, Abcam), rabbit anti-human LRG1 (1:200, HPA001888, Atlas Antibodies, Bromma, Sweden), mouse anti-human GJA5 (1:250, clone 2F9A11, 37-8900, Invitrogen), mouse anti-human ASS1 (1:100, clone 2B10, ab124465, Abcam), rabbit anti-human S100A4 (1:200, ab41532, Abcam), goat anti-human SOX17 (1:10, AF1924, R&D) and mouse anti-human PLAUR (1:100, clone 62022, MAB807, R&D).

Techniques: Marker, Expressing

Journal: Kidney international

Article Title: Connexins 40 and 43 are differentially regulated within the kidneys of rats with renovascular hypertension.

doi: 10.1046/j.1523-1755.2001.00786.x

Figure Lengend Snippet: Fig. 2. Antibodies against Cx40 specifically recognize gap junctions. (A) The antibodies to Cx40 that were used throughout this study did not stain the internal elastic lamina and smooth muscle cells (SMC) of a rat aorta. In contrast, the protein A-gold particles that were reacted with the antibodies selectively decorated minute areas of apposition (arrows) between the membranes of adjacent endothe- lial cells (EC). (B) At higher magnification, the area of membrane apposition pointed by the arrows in (A) features the pentalaminar appearance characteristic of a gap junction plaque and is decorated by 15 nm gold parti- cles, reflecting the presence of Cx40. (C) Im- munofluorescence labeling with the same anti- bodies located Cx40 at discrete spots along the endothelial layer of the rat aortic wall but not over the media. Note that the endothelial labeling was interrupted at sites of cell nuclei (arrow heads). (D) is the phase contrast view of the field shown in (C). Bar represents 3 m in (A), 1.1 m in (B), and 70 m in (C and D). L, lumen of vessel.

Article Snippet: Sections were rinsed in PBS, incubated for 30 minuteswith a Kinametic polytron blender (Kriens, Switzerland) in 100 mmol/L Tris-HCl (pH 7.4), supplemented with in a buffer containing 0.5% BSA, and then exposed for 20 hours to antibodies against either Cx40 (diluted 1:40020 mmol/L ethylenediaminetetraacetic acid (EDTA), 1 g/mL pepstatin A, 1 g/mL antipain (Merck, Switzer- in PBS), Cx43 (diluted 1:500 in PBS; Zymed Laboratories Inc.), or renin (diluted 1:200 in PBS).

Techniques: Staining, Membrane, Labeling

Journal: Kidney international

Article Title: Connexins 40 and 43 are differentially regulated within the kidneys of rats with renovascular hypertension.

doi: 10.1046/j.1523-1755.2001.00786.x

Figure Lengend Snippet: Fig. 3. Distribution of Cx40 mRNA in the kidney. In situ hybridization using two differ- ent antisense rat cDNA probes specific for Cx40 demonstrated that the mRNA coding for this protein is expressed in the endothelium of small kidney vessels (A), including juxtaglom- erular arterioles (arrow heads in C and D) and glomerular capillaries (C and D). No sig- nal was detected when sections of the very same kidney were hybridized with a sense probe (B). Bar represents 100 m.

Article Snippet: Sections were rinsed in PBS, incubated for 30 minuteswith a Kinametic polytron blender (Kriens, Switzerland) in 100 mmol/L Tris-HCl (pH 7.4), supplemented with in a buffer containing 0.5% BSA, and then exposed for 20 hours to antibodies against either Cx40 (diluted 1:40020 mmol/L ethylenediaminetetraacetic acid (EDTA), 1 g/mL pepstatin A, 1 g/mL antipain (Merck, Switzer- in PBS), Cx43 (diluted 1:500 in PBS; Zymed Laboratories Inc.), or renin (diluted 1:200 in PBS).

Techniques: In Situ Hybridization

Journal: Kidney international

Article Title: Connexins 40 and 43 are differentially regulated within the kidneys of rats with renovascular hypertension.

doi: 10.1046/j.1523-1755.2001.00786.x

Figure Lengend Snippet: Fig. 4. Cx40 and Cx43 are differentially dis- tributed in the kidney. Specific antibodies against the carboxy terminus of Cx40 showed abundant levels of this protein along the endo- thelium of small kidney vessels (A) and be- tween media cells of the afferent arteriole (ar- rowhead in B and C). Much less Cx40 was also seen between the mesangial and endothe- lial cells of glomeruli (B). (C) is the phase contrast of the field shown in (B). Antibodies to Cx43 showed the presence of this connexin between the endothelial cells of medium size (D) and small kidney arterioles, including the afferent arteriole (arrows in E). In contrast, Cx43 was not found between the media cells of the latter vessel (arrowhead in E) and was barely visible within glomeruli (E). (F) The phase contrast view of the field shown in (E). The bar represents 15 m.

Article Snippet: Sections were rinsed in PBS, incubated for 30 minuteswith a Kinametic polytron blender (Kriens, Switzerland) in 100 mmol/L Tris-HCl (pH 7.4), supplemented with in a buffer containing 0.5% BSA, and then exposed for 20 hours to antibodies against either Cx40 (diluted 1:40020 mmol/L ethylenediaminetetraacetic acid (EDTA), 1 g/mL pepstatin A, 1 g/mL antipain (Merck, Switzer- in PBS), Cx43 (diluted 1:500 in PBS; Zymed Laboratories Inc.), or renin (diluted 1:200 in PBS).

Techniques:

Journal: Kidney international

Article Title: Connexins 40 and 43 are differentially regulated within the kidneys of rats with renovascular hypertension.

doi: 10.1046/j.1523-1755.2001.00786.x

Figure Lengend Snippet: Fig. 5. Cx40 is expressed in the portion of the afferent arteriole that comprises renin-secreting cells. Immunolabeling of the cortex of a rat kidney showed that Cx40, as revealed using a fluorescein-tagged secondary antibody, was abundant in the juxtaglomerular portion of the afferent arteriole (A), which was also positive for renin-containing cells, as revealed using a rhodamin-tagged secondary antibody (B). Double exposure of the same area showed that most of the Cx40 signal colocalized with that of renin, resulting in a yellow staining of the juxtaglomerular portion of the afferent arteriole (C). Under similar conditions, the antibody to Cx40 usually elicited a more extensive staining in the afferent arteriole of hypertensive (E) than normotensive rats (D). The bar represents 22 m in (A–C) and 11 m in (D and E).

Article Snippet: Sections were rinsed in PBS, incubated for 30 minuteswith a Kinametic polytron blender (Kriens, Switzerland) in 100 mmol/L Tris-HCl (pH 7.4), supplemented with in a buffer containing 0.5% BSA, and then exposed for 20 hours to antibodies against either Cx40 (diluted 1:40020 mmol/L ethylenediaminetetraacetic acid (EDTA), 1 g/mL pepstatin A, 1 g/mL antipain (Merck, Switzer- in PBS), Cx43 (diluted 1:500 in PBS; Zymed Laboratories Inc.), or renin (diluted 1:200 in PBS).

Techniques: Immunolabeling, Staining

Journal: Kidney international

Article Title: Connexins 40 and 43 are differentially regulated within the kidneys of rats with renovascular hypertension.

doi: 10.1046/j.1523-1755.2001.00786.x

Figure Lengend Snippet: Fig. 6. Cx40 links renin-containing cells, en- dothelial cells, as well as renin and endothelial cells of the afferent arteriole. The ultrastruc- tural distribution of Cx40 was studied in sec- tions of the juxtaglomerular portion (gc, glo- merular capillary) of afferent arterioles (aa). (A) As judged by the decoration of the protein A-gold particles that were reacted with the anti- bodies to Cx40, this connexin was selectively located at minute areas of apposition between the membranes of renin-containing (rc; rect- angle 1) and endothelial cells (ec; rectangle 2). (B and C) Higher magnification views of the fields outlined by rectangles 1 and 2 in (A). (D) Renin-containing cells were identified by their characteristic secretory granules, which were decorated by protein A-gold particles when reacted with antirenin antibodies. (E) Cx40 was also found at contacts between the membranes of renin-containing and endothe- lial cells (rectangle 3). (F) Higher magnifica- tion view of the field outlined by rectangle 3 in (E). (G and H) when the orientation of sectioning was favorable, the areas immunola- beled for Cx40 between endothelial cells (G) and between renin-containing cells (H) dis- played the pentalaminar appearance of mem- brane appositions (the arrowheads point to the middle dark line), characteristic of gap junctions in aldehyde-fixed tissues. The bar represents 1.5 m in A; 150 nm in B, C, and F; 500 nm in D and E; 125 nm in G; and 112 nm in H.

Article Snippet: Sections were rinsed in PBS, incubated for 30 minuteswith a Kinametic polytron blender (Kriens, Switzerland) in 100 mmol/L Tris-HCl (pH 7.4), supplemented with in a buffer containing 0.5% BSA, and then exposed for 20 hours to antibodies against either Cx40 (diluted 1:40020 mmol/L ethylenediaminetetraacetic acid (EDTA), 1 g/mL pepstatin A, 1 g/mL antipain (Merck, Switzer- in PBS), Cx43 (diluted 1:500 in PBS; Zymed Laboratories Inc.), or renin (diluted 1:200 in PBS).

Techniques:

Journal: Kidney international

Article Title: Connexins 40 and 43 are differentially regulated within the kidneys of rats with renovascular hypertension.

doi: 10.1046/j.1523-1755.2001.00786.x

Figure Lengend Snippet: Fig. 7. Expression of Cx40, Cx43, and renin genes in kidneys of normotensive and hyper- tensive rats. (A and B) Northern blots re- vealed that compared with the levels normal- ized to the GAPDH signal observed in sham- operated animals, the levels of the Cx40 tran- script were increased in both left (LK) and right (RK) kidneys of 2K1C rats, whereas those of the Cx43 transcript were increased only in the unclipped right kidney (RK). The blots also show that the expression of the renin gene was markedly enhanced in the clipped left kidney (LK) and decreased in the unclipped right kidney of the hypertensive 2K1C rats. In the very same samples, levels of GAPDH mRNA were not modified. In left panels, each lane shows a sample of left kidney from a different rat. In right panels, corresponding lanes show samples of right kidneys of the very same animals. All lanes were loaded with 6 g poly A mRNA. (C) Quantitative assess- ment of nine experiments (one per rat) con- firmed that the levels of Cx40 mRNA were increased (P 0.01) approximately twofold over control values in both left (LK) and right (RK) of 2K1C rats. In contrast, those of Cx43 were significantly increased only in the right unclipped kidney of 2K1C rats. Values repre- sent ratios of densitometric measurements of either Cx40 or Cx43 and GAPDH mRNAs and are expressed as mean SEM. *P 0.05; **P 0.01 level.

Article Snippet: Sections were rinsed in PBS, incubated for 30 minuteswith a Kinametic polytron blender (Kriens, Switzerland) in 100 mmol/L Tris-HCl (pH 7.4), supplemented with in a buffer containing 0.5% BSA, and then exposed for 20 hours to antibodies against either Cx40 (diluted 1:40020 mmol/L ethylenediaminetetraacetic acid (EDTA), 1 g/mL pepstatin A, 1 g/mL antipain (Merck, Switzer- in PBS), Cx43 (diluted 1:500 in PBS; Zymed Laboratories Inc.), or renin (diluted 1:200 in PBS).

Techniques: Expressing, Northern Blot, Control

Journal: Kidney international

Article Title: Connexins 40 and 43 are differentially regulated within the kidneys of rats with renovascular hypertension.

doi: 10.1046/j.1523-1755.2001.00786.x

Figure Lengend Snippet: Fig. 8. Expression of the Cx40 gene in aortic endothelial cells of 2K1C hypertensive rats. (A) Northern blots revealed that the levels of Cx40 mRNA were higher in the aorta of hypertensive 2K1C rats than in that of sham-operated, normotensive controls. Each lane is from a different animal and was loaded with 15 g RNA. (B) Quantitative densitometric analysis of eight independent experiments (one per rat) showed that relative to the levels of GAPDH mRNA that were constant, those of Cx40 mRNA were twice as high in 2K1C than in sham-operated rats. *P 0.01.

Article Snippet: Sections were rinsed in PBS, incubated for 30 minuteswith a Kinametic polytron blender (Kriens, Switzerland) in 100 mmol/L Tris-HCl (pH 7.4), supplemented with in a buffer containing 0.5% BSA, and then exposed for 20 hours to antibodies against either Cx40 (diluted 1:40020 mmol/L ethylenediaminetetraacetic acid (EDTA), 1 g/mL pepstatin A, 1 g/mL antipain (Merck, Switzer- in PBS), Cx43 (diluted 1:500 in PBS; Zymed Laboratories Inc.), or renin (diluted 1:200 in PBS).

Techniques: Expressing, Northern Blot