primary antibody against serca2 atpase  (Thermo Fisher)

Journal: International Journal of Molecular Sciences

Article Title: Ryanodine Receptor Staining Identifies Viable Cardiomyocytes in Human and Rabbit Cardiac Tissue Slices

doi: 10.3390/ijms241713514

Figure Lengend Snippet: Methodological approach. ( A ) Human and rabbit myocardial slices were created from left-ventricular transmural samples with a vibratome and then cultivated for up to seven days in biomimetic cultivation chambers containing stimulation electrodes and a force transducer. Subsequently, dextran-based death staining was applied, followed by chemical fixation, fluorescent staining, confocal microscopy and image processing. ( B ) Example image of a rabbit cardiac slice stained with fixable, fluorescent dextran (green), for ryanodine receptors (RyR, red) and with wheat germ agglutinin (WGA, blue). ( C ) Histogram-based local thresholds were applied to identify the dextran-positive (green) and RyR-positive (red) pixels, as well as ( D ) the WGA-positive pixels (white). ( E ) Using the WGA distance map, a watershed transform was applied to segment individual myocytes (different colors). ( F ) For each cell segment, the number of RyR- and dextran-positive pixels was counted. If the fraction of these pixels within the segment exceeded a defined threshold, segments were classified as RyR-positive, i.e., living, myocytes (magenta) and dextran-positive, i.e., dead, myocytes (green). Scale bar length in ( B ) is 20 µm and also applies to ( C – F ).

Article Snippet: For immunostaining, the slices were incubated with primary antibody against cardiac ryanodine receptor (IgG1, mouse, C3-33, Thermo Fisher, Braunschweig, Germany) or SERCA2 (IgG2a, mouse, 2A7-A1, Thermo Fisher) 1:200 in blocking solution (BS: 5% NGS, 5% BSA, 0.25% Triton-X in PBS) for 4 h at RT or overnight at 4 °C.

Techniques: Staining, Confocal Microscopy

Journal: British Journal of Pharmacology

Article Title: Inactivation of Cys 674 in SERCA2 increases BP by inducing endoplasmic reticulum stress and soluble epoxide hydrolase

doi: 10.1111/bph.14937

Figure Lengend Snippet: Inactivation of SERCA2 C674 increases BP and decreases urine output and sodium excretion. (a) BP. * P < .05, significantly different as indicated, n = 10. (b) 24‐hr urine volume and urine sodium excretion, * P < .05, significantly different as indicated, n = 8. (c) Na+/K+‐ATPase activity in primary RPT cells. * P < .05, significantly different as indicated, n = 7. All data are presented as means ± SEM, unpaired Student's t test

Article Snippet: Primary antibodies against SERCA2 C674‐SO 3 H (Ying et al., 2008 ; C674‐SO 3 H antibody was a custom polyclonal antibody from Bethyl Laboratories, Inc.), https://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=2970 ( https://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=2970 ; Santa Cruz, Cat# sc‐87099, RRID:AB_2293440), or https://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=214 (Abcam, Cat# ab81296, RRID:AB_2814742) were incubated overnight at 4°C according to the staining procedure of HRP‐DAB kit (CWBIO, Cat# CW0125M).

Techniques: Activity Assay

Journal: British Journal of Pharmacology

Article Title: Inactivation of Cys 674 in SERCA2 increases BP by inducing endoplasmic reticulum stress and soluble epoxide hydrolase

doi: 10.1111/bph.14937

Figure Lengend Snippet: Inactivation of SERCA2 C674 increases intracellular Ca2+ levels in RPT cells. (a) The mRNA levels of both SERCA2a and SERCA2b in the renal cortex. n = 5. (b) SERCA2 protein levels in the renal cortex and primary RPT cells. n = 8. (c) Intracellular Ca2+ detected by Fluo‐4 in primary RPT cells. * P < .05, significantly different as indicated, n = 6. All data are presented as mean ± SEM, unpaired Student's t test

Article Snippet: Primary antibodies against SERCA2 C674‐SO 3 H (Ying et al., 2008 ; C674‐SO 3 H antibody was a custom polyclonal antibody from Bethyl Laboratories, Inc.), https://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=2970 ( https://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=2970 ; Santa Cruz, Cat# sc‐87099, RRID:AB_2293440), or https://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=214 (Abcam, Cat# ab81296, RRID:AB_2814742) were incubated overnight at 4°C according to the staining procedure of HRP‐DAB kit (CWBIO, Cat# CW0125M).

Techniques:

Journal: British Journal of Pharmacology

Article Title: Inactivation of Cys 674 in SERCA2 increases BP by inducing endoplasmic reticulum stress and soluble epoxide hydrolase

doi: 10.1111/bph.14937

Figure Lengend Snippet: The sustained induction of ER stress by inactivation of SERCA2 C674 accounts for the elevated BP. (a) Representative Western blots of ER stress markers from the renal cortex and quantification of band intensities in graph. Mean ± SEM, unpaired Student's t test, * P < .05, significantly different as indicated, n = 5. (b) Representative Western blots of ER stress markers from primary RPT cells and quantification of band intensities in graph. Mean ± SEM, unpaired Student's t test, * P < .05, significantly different as indicated, p‐PERK, n = 7; BIP, n = 6; ATF6, n = 5; CHOP, n = 6. (c) Na+/K+‐ATPase activity in primary SKI RPT cells treated with 4‐PBA. Mean ± SEM, unpaired Student's t test, * P < .05, significantly different as indicated, n = 5. (d) The effects of 4‐PBA on BP. Mean ± SEM, two‐way ANOVA, * P < .05, significantly different as indicated,; # P < .05, significantly different as indicated, n = 5. (e) The effects of 4‐PBA on SKI urine volume and urine sodium secretion. Mean ± SEM, unpaired Student's t test, * P < .05, significantly different as indicated, n = 5

Article Snippet: Primary antibodies against SERCA2 C674‐SO 3 H (Ying et al., 2008 ; C674‐SO 3 H antibody was a custom polyclonal antibody from Bethyl Laboratories, Inc.), https://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=2970 ( https://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=2970 ; Santa Cruz, Cat# sc‐87099, RRID:AB_2293440), or https://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=214 (Abcam, Cat# ab81296, RRID:AB_2814742) were incubated overnight at 4°C according to the staining procedure of HRP‐DAB kit (CWBIO, Cat# CW0125M).

Techniques: Western Blot, Activity Assay

Journal: British Journal of Pharmacology

Article Title: Inactivation of Cys 674 in SERCA2 increases BP by inducing endoplasmic reticulum stress and soluble epoxide hydrolase

doi: 10.1111/bph.14937

Figure Lengend Snippet: Inactivation of SERCA2 C674 up‐regulates sEH expression and down‐regulates expression of D1 receptors. (a) Representative Western blots of sEH and D1 receptors from the renal cortex and quantification of band intensities in graph. * P < .05, significantly different as indicated, sEH, n = 7; D1 receptors, n = 5. (b) Representative Western blots of sEH and D1 receptors from primary RPT cells and quantification of band intensities in graph. * P < .05, significantly different as indicated, sEH, n = 7; D1 receptors, n = 8. (c) Representative picture of immunofluorescence of sEH and D1 receptors in primary RPT cells. Quantification in graph, * P < .05, significantly different as indicated, n = 5. Scale bars = 75 μm. (d) Representative picture of immunohistochemical staining of sEH and D1 receptors in frozen sections of kidney. Quantification in graph, * P < .05, significantly different as indicated, n = 7. Scale bars = 200 μm. All data are presented as mean ± SEM, unpaired Student's t test

Article Snippet: Primary antibodies against SERCA2 C674‐SO 3 H (Ying et al., 2008 ; C674‐SO 3 H antibody was a custom polyclonal antibody from Bethyl Laboratories, Inc.), https://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=2970 ( https://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=2970 ; Santa Cruz, Cat# sc‐87099, RRID:AB_2293440), or https://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=214 (Abcam, Cat# ab81296, RRID:AB_2814742) were incubated overnight at 4°C according to the staining procedure of HRP‐DAB kit (CWBIO, Cat# CW0125M).

Techniques: Expressing, Western Blot, Immunofluorescence, Immunohistochemical staining, Staining