serca2 primary antibody  (Novus Biologicals)

Journal: bioRxiv

Article Title: Deciphering the functional association of novel variants of BMP7 in isolated congenital heart disease by integrating in vitro and in silico approaches

doi: 10.64898/2025.12.23.696272

Figure Lengend Snippet: Expression analysis of BMP7 wild-type (WT) and muteins (MUT) by immunostaining and immunoblotting and colocalization of BMP7 proteins with SERCA2 and ER-Tracker. (A1-F4) Immunostaining in H9c2 cells showing cellular location of BMP7 WT and MUT (p.D85V, p.R175W, p.A283T, p.M315I and p.N321S). (A5-F8) Immunostaining in H9c2 cells showing colocalization of BMP7 proteins with SERCA2 with quantitative assessment by Scatter plot. (A9-A12) Immunostaining in H9c2 cells showing colocalization of BMP7 proteins with ER-Tracker with quantitative assessment by Scatter plot. (A13-F13) EdU staining showing cell proliferation due to all the variants in H9c2 cells. (G) Western blot analysis of WT and MUT in P19 cells showing expression of BMP7 and pSMAD1/5 due to WT and all five variants (p.D85V, p.R175W, p.A283T, p.M315I and p.N321S) with graphical representation in terms of fold change for pSmad1/5. (H) Western blotting showing expression level of pSMAD1/5 due to WT and all five variants (p.D85V, p.R175W, p.A283T, p.M315I and p.N321S) when co-transfected with ALK2.

Article Snippet: Then the cells were incubated with diluted (1:100) SERCA2 primary antibody (Novus Biological, USA) overnight at 4°C.

Techniques: Expressing, Immunostaining, Western Blot, Staining, Transfection

Journal: Cell Reports Medicine

Article Title: Endoplasmic reticulum stress-related deficits in calcium clearance promote neuronal dysfunction that is prevented by SERCA2 gene augmentation

doi: 10.1016/j.xcrm.2024.101839

Figure Lengend Snippet: OHT leads to ER stress and SERCA2 downregulation (A–D) Representative flow cytometry histograms of pPERK (A), peIF2α (B), ATF4 (C), or CHOP (D) in RGCs of sham and OHT retinas. Flow cytometry quantification demonstrated increased pPERK, peIF2α, ATF4, or CHOP protein expression (mean fluorescence intensity: MFI) in glaucomatous RGCs relative to sham controls (sham: N = 4 mice/group, OHT: N = 4 mice/group, two-tailed Student’s t test, ∗∗ p < 0.01, ∗ p < 0.05). A non-targeting isotype antibody was used as a non-specific control. (E and F) Quantitative RT-PCR-based gene profiling shows reduced ATP2A2 mRNA levels in whole retinas (E) or FAC-sorted RGCs (F) at 2 weeks after OHT induction (sham: N = 3–7 mice/group, OHT: N = 4–9 mice/group; two-tailed Student’s t test, ∗ p < 0.05). (G and H) Representative flow cytometry histogram and MFI of SERCA2 in RGCs from sham and OHT retinas (sham: N = 4 mice, OHT: N = 3 mice, two-tailed Student’s t test, ∗ p < 0.05). (I and J) Immunohistochemical analysis of mouse retinal cross-sections using antibodies against SERCA2, the RGC-specific marker RBPMS, and the ER marker calreticulin confirms a significant decrease in SERCA2 expression within the ER in glaucomatous RGCs. (K) Quantification of epifluorescence intensity in SERCA2-positive RGCs demonstrates a decrease in SERCA2 activity in glaucomatous retinas relative to sham control (sham: N = 5 mice, n = 144 RGCs; OHT: N = 5 mice, n = 159 RGCs; two-tailed Student’s t test, ∗∗∗ p < 0.001). (L and M) SERCA2 immunostaining in retinas from patients with primary open-angle glaucoma and age-matched controls reveals reduced SERCA2 protein in RGCs, visualized with RBPMS. (N) Quantification of epifluorescence intensity in SERCA2-positive RGCs revealed reduced SERCA2 protein levels in glaucomatous retinas relative to age-matched controls (controls: N = 8 human retinas, n = 151 RGCs; glaucoma: N = 13 human retinas, n = 176 RGCs; two-tailed Student’s t test, ∗∗∗ p < 0.001, Table S1 ).

Article Snippet: Paraffin retinal sections were heated for 20 min in citrate buffer (80°C–90°C) followed by incubation in blocking solution (10% normal donkey serum, 1% bovine serum albumin, 0.5% Triton X-100) for 1 h. Retinal sections were incubated with primary antibodies: SERCA2 (4 μg/mL, Invitrogen), Calreticulin (1:250, Invitrogen), and RBPMS (0.25 μg/mL, PhosphoSolutions), followed by anti-mouse Alexa 647, anti-rabbit Alexa Fluor 594, or anti-guinea pig Alexa Fluor 488 (2–4 μg/mL, Molecular Probes).

Techniques: Flow Cytometry, Expressing, Fluorescence, Two Tailed Test, Control, Quantitative RT-PCR, Immunohistochemical staining, Marker, Activity Assay, Immunostaining

Journal: Cell Reports Medicine

Article Title: Endoplasmic reticulum stress-related deficits in calcium clearance promote neuronal dysfunction that is prevented by SERCA2 gene augmentation

doi: 10.1016/j.xcrm.2024.101839

Figure Lengend Snippet: SERCA2 downregulation is consistent with Ca 2+ clearance deficits, and pharmacological activation of SERCA2 restores Ca 2+ dynamics and promotes neuronal survival (A) Representative traces of light-evoked Ca 2+ responses of single RGCs in naive Thy1-GCaMP6f retinas before and after treatment with cyclopiazonic acid (CPA), an SERCA inhibitor. Exponential fits show increased decay time constant (τ) with CPA. (B–D) Quantitative analysis of longitudinal light-evoked Ca 2+ responses in the same RGC before and after treatment with CPA shows that Ca 2+ decay time is substantially delayed by CPA, while other parameters are not affected ( N = 7 mice, n = 9 RGCs; Wilcoxon matched-pairs signed rank test, ∗∗ p < 0.01, n.s.: not significant). (E–H) Treatment with vehicle (PBS) did not alter light-evoked Ca 2+ responses validating our approach ( N = 3 mice, n = 4 RGCs; Wilcoxon matched-pairs signed rank test, n.s.: not significant). (I) Representative traces of light-evoked RGC Ca 2+ responses in OHT-induced Thy1-GCaMP6f retinas before and after CDN1163 treatment, an SERCA activator. (J–L) Quantitative analysis of responses from the same neuron before and after treatment with CDN1163 shows that SERCA activation rescues Ca 2+ dynamics ( N = 5 mice, n = 10 RGCs; Wilcoxon matched-pairs signed rank test, ∗∗ p < 0.01, n.s.: not significant). (M) Representative traces of light-evoked Ca 2+ responses of single RGCs in OHT-induced Thy1-GCaMP6f retinas before and after PBS control treatment. (N–P) Quantitative analysis of responses in the same glaucomatous RGC soma before and after treatment with PBS shows that PBS has no effect on light-evoked Ca 2+ dynamics ( N = 5 mice, n = 11 RGCs; Wilcoxon matched-pairs signed rank test, n.s.: not significant). (Q–S) Representative images of RBPMS-labeled flat-mounted retinas from sham (Q), and OHT eyes treated with PBS (R) or CDN1163 (S). (T) Quantification of RBPMS-positive RGCs demonstrates that CDN1163 promotes potent neuroprotection at 3 weeks of OHT induction (sham: N = 4 mice, OHT+PBS: N = 6 mice, OHT+CDN1163: N = 6 mice, one-way ANOVA with Tukey’s multiple comparisons post hoc test, ∗∗∗ p < 0.001). Data are presented as mean values ± SEM.

Article Snippet: Paraffin retinal sections were heated for 20 min in citrate buffer (80°C–90°C) followed by incubation in blocking solution (10% normal donkey serum, 1% bovine serum albumin, 0.5% Triton X-100) for 1 h. Retinal sections were incubated with primary antibodies: SERCA2 (4 μg/mL, Invitrogen), Calreticulin (1:250, Invitrogen), and RBPMS (0.25 μg/mL, PhosphoSolutions), followed by anti-mouse Alexa 647, anti-rabbit Alexa Fluor 594, or anti-guinea pig Alexa Fluor 488 (2–4 μg/mL, Molecular Probes).

Techniques: Activation Assay, Control, Labeling

Journal: Cell Reports Medicine

Article Title: Endoplasmic reticulum stress-related deficits in calcium clearance promote neuronal dysfunction that is prevented by SERCA2 gene augmentation

doi: 10.1016/j.xcrm.2024.101839

Figure Lengend Snippet: SERCA2 activation restores Ca 2+ dynamics and promotes RGC survival after traumatic optic nerve injury (A and B) Schematic of the mouse optic nerve crush (ONC) model, which does not affect intraocular pressure (IOP) ( N = 12 mice/group, two-way ANOVA with Sidak’s multiple comparison post hoc test, n.s.: not significant). (C) Axonal injury leads to significant RGC loss starting at 1 week after ONC (sham: N = 5 mice/group; one-way ANOVA with Dunnett’s post hoc test, ∗∗∗ p < 0.001, n.s.: not significant). (D and E) Immunohistochemical analysis of retinal cross-sections with antibodies against SERCA2, RBPMS, and calreticulin confirms a significant reduction in SERCA2 expression in the ER of injured RGCs. (F) Quantification of epifluorescence intensity in SERCA2-positive RGCs demonstrates a decrease in SERCA2 activity at 4 days post ONC relative to sham controls (sham: N = 3 mice, n = 51 RGCs; ONC: N = 4 mice, n = 66 RGCs; two-tailed Student’s t test, ∗∗∗ p < 0.001). (G) In naive (uninjured) RGC soma, light stimulation elicits a brief Ca 2+ transient with fast signal decay. Exponential fits show the decay time constant (τ). (H) Injured ON-RGCs from ONC eyes display slower Ca 2+ responses characterized by increased signal decay time. (I–K) Quantitative analysis of Ca 2+ signal decay (I), rise rate (J), and amplitude (K) from sham and ONC retinas (sham: N = 6 mice, n = 55 RGCs; ONC: N = 6 mice, n = 38 RGCs; two-tailed Welch’s t test, ∗ p < 0.05, n.s.: not significant). (L) Representative traces of light-evoked RGC Ca 2+ responses from ONC Thy1-GCaMP6f retinas before and after treatment with the SERCA activator CDN1163. (M–O) Responses from the same neuron before and after treatment with CDN1163 show that SERCA activation rescues Ca 2+ dynamics after ONC ( N = 5 mice, n = 10 RGCs; Wilcoxon matched-pairs signed rank test, ∗ p < 0.01, n.s.: not significant). (P) Representative traces of light-evoked Ca 2+ responses of single RGCs in Thy1-GCaMP6f retinas with ONC before and after PBS control treatment. (Q–S) Longitudinal analysis of responses from the same injured RGC before and PBS administration shows no change in light-evoked Ca 2+ dynamics ( N = 5 mice, n = 11 RGCs; Wilcoxon matched-pairs signed rank test, n.s.: not significant). (T–W) Representative images of RBPMS-labeled flat-mounted retinas from sham (T), and ONC eyes treated with PBS (U) or CDN1163 (V). (T) Quantification of RBPMS-positive RGCs shows that CDN1163 promotes RGC neuroprotection at 1 week of ONC ( N = 5 mice/group, one-way ANOVA with Tukey’s multiple comparisons post hoc test, ∗∗∗ p < 0.001). Data are presented as mean values ± SEM. The cartoon in this figure was generated with BioRender ( https://biorender.com ).

Article Snippet: Paraffin retinal sections were heated for 20 min in citrate buffer (80°C–90°C) followed by incubation in blocking solution (10% normal donkey serum, 1% bovine serum albumin, 0.5% Triton X-100) for 1 h. Retinal sections were incubated with primary antibodies: SERCA2 (4 μg/mL, Invitrogen), Calreticulin (1:250, Invitrogen), and RBPMS (0.25 μg/mL, PhosphoSolutions), followed by anti-mouse Alexa 647, anti-rabbit Alexa Fluor 594, or anti-guinea pig Alexa Fluor 488 (2–4 μg/mL, Molecular Probes).

Techniques: Activation Assay, Comparison, Immunohistochemical staining, Expressing, Activity Assay, Two Tailed Test, Control, Labeling, Generated

Journal: Cell Reports Medicine

Article Title: Endoplasmic reticulum stress-related deficits in calcium clearance promote neuronal dysfunction that is prevented by SERCA2 gene augmentation

doi: 10.1016/j.xcrm.2024.101839

Figure Lengend Snippet: SERCA2 gene supplementation rescues Ca 2+ dynamics during OHT- and ONC-induced stress (A) Confocal micrographs show co-localization of AAV-mediated SERCA2 and RBPMS in RGC soma. (A′) Higher magnification images show AAV-driven SERCA2 expression in RGCs. (B) Cross-section from retinas transduced with AAV. SERCA2 shows RGC-specific expression. Nuclei stained with DAPI. (C) Quantification of the number of RGCs transfected by AAV.SERCA2 shows that ∼50% of all RBPMS-positive RGCs co-expressed AAV-mediated SERCA2. (D) Representative flow cytometric histograms and MFI of SERCA2 in RGCs from AAV.Ctl- and AAV.SERCA2-treated retinas. Quantitative analysis shows increased SERCA2 protein expression in AAV.SERCA2-infected RGCs (mCherry + ) relative to control AAV (AAV.Ctl: N = 5 retinas, AAV.SERCA2: N = 6 retinas, two-tailed Student’s t test, ∗∗∗ p < 0.001). A non-targeting isotype antibody was used as a non-specific control. (E–G) Time-lapse TPLSM recordings of ON-RGCs from sham uninjured Thy1-GCaMP6f retinas (E) and OHT Thy1-GCaMP6f eyes treated with AAV.Ctl (F) or AAV.SERCA2 (G). (H) In uninjured RGCs from sham retinas, light stimulation elicits a brief Ca 2+ transient with fast signal decay. Exponential fits show the decay time constant (τ). (I) Injured ON-RGCs from AAV.Ctl-treated OHT eyes display slower Ca 2+ responses, notably longer signal decay. (J) AAV.SERCA2 treatment restored light-evoked Ca 2+ dynamics to values like those in uninjured sham eyes. (K–M) Quantitative analysis confirmed that AAV.SERCA2 substantially reduced Ca 2+ decay time during OHT relative to AAV.Ctl (sham: N = 8 mice, n = 61 RGCs; OHT+AAV.Ctl: N = 16 mice, n = 81 RGCs; OHT+AAV.SERCA2: N = 15 mice, n = 65 RGCs; Kruskal-Wallis test, ∗∗∗ p < 0.001, ∗ p < 0.05, n.s.: not significant). (N–P) Representative Ca 2+ signal traces from Thy1-GCaMP6f retinal explants of naive (sham) RGCs (N) and glaucomatous retinas with AAV.Ctl (O) or AAV.SERCA2 (P). (Q–S) Quantitative analysis confirmed that AAV.SERCA2 treatment improved Ca 2+ dynamics recorded from retinal explants (sham: N = 5 mice, n = 501 RGCs; OHT+AAV.Ctl: N = 8 mice, n = 213 RGCs; OHT+AAV.SERCA2: N = 8 mice, n = 102 RGCs soma; Kruskal-Wallis test, ∗∗∗ p < 0.001, n.s.: not significant). (T and U) The ON-OFF index calculated in AAV.Ctl-treated glaucomatous retinas shifts toward the ON response (T). In contrast, restoration of the ON-OFF index is observed in AAV.SERCA2-treated injured RGCs (U) (sham mice: N = 5 mice, n = 501 RGCs; OHT+AAV.Ctl mice: N = 8 mice, n = 213 RGCs; OHT+AAV.SERCA2 mice: N = 8 mice, n = 102 RGCs; two-tailed Student’s t test, ∗ p < 0.05, n.s.: not significant). (V–X) Representative TPLSM Ca 2+ signal traces of RGCs in sham controls (V) and at 1 week after ONC treated with AAV.Ctl (W) or AAV.SERCA2 (X). (Y–AA) Quantitative analysis confirmed that AAV.SERCA2 substantially reduced Ca 2+ decay time after ONC relative to AAV.Ctl (sham: N = 6 mice, n = 55 RGCs; ONC+AAV.Ctl: N = 6 mice, n = 59 RGCs; ONC+AAV.SERCA2: N = 6 mice, n = 40 RGCs; Kruskal-Wallis test, ∗∗∗ p < 0.001, n.s.: not significant). Data are presented as mean values ± SEM.

Article Snippet: Paraffin retinal sections were heated for 20 min in citrate buffer (80°C–90°C) followed by incubation in blocking solution (10% normal donkey serum, 1% bovine serum albumin, 0.5% Triton X-100) for 1 h. Retinal sections were incubated with primary antibodies: SERCA2 (4 μg/mL, Invitrogen), Calreticulin (1:250, Invitrogen), and RBPMS (0.25 μg/mL, PhosphoSolutions), followed by anti-mouse Alexa 647, anti-rabbit Alexa Fluor 594, or anti-guinea pig Alexa Fluor 488 (2–4 μg/mL, Molecular Probes).

Techniques: Expressing, Transduction, Staining, Transfection, Infection, Control, Two Tailed Test

Journal: Cell Reports Medicine

Article Title: Endoplasmic reticulum stress-related deficits in calcium clearance promote neuronal dysfunction that is prevented by SERCA2 gene augmentation

doi: 10.1016/j.xcrm.2024.101839

Figure Lengend Snippet: SERCA2 gene therapy attenuates ER stress, promotes neuronal survival, and prevents loss of visual function (A–D) Representative flow cytometry histograms show that AAV.SERCA2 reduced the expression of the ER stress markers pPERK, peIF2α, ATF4, and CHOP during OHT relative to AAV.Ctl (OHT+AAV.Ctl: N = 6 retinas, OHT+AAV.SERCA2: N = 6 retinas, two-tailed Student’s t test, ∗∗∗ p < 0.001, ∗∗ p < 0.01, ∗ p < 0.05). A non-targeting isotype antibody was used as a non-specific control. (E–G) Representative images of RBPMS-labeled flat-mounted retinas from sham (E) and 3 weeks of OHT treated with AAV.Ctl (F) or AAV.SERCA2 (G). (H) Quantification of RGC density shows that AAV.SERCA2 promotes neuronal survival at 3 and 4 weeks of OHT induction relative to control eyes (sham: N = 4 mice, OHT+AAV.Ctl: N = 6–8 mice, OHT+AAV.SERCA2: N = 5–7 mice; one-way ANOVA with Tukey’s multiple comparisons post hoc test, ∗∗∗ p < 0.001). (I–K) Representative images of RBPMS-labeled flat-mounted retinas from sham (I) and at 2 weeks of ONC eyes treated with AAV.Ctl (J) or AAV.SERCA2 (K). (L) Quantification of RGC density shows that AAV.SERCA2 promotes neuronal survival relative to control eyes at 1 and 2 weeks after ONC injury ( N = 5/group; one-way ANOVA with Tukey’s multiple comparisons post hoc test, ∗∗∗ p < 0.001). (M) Optomotor reflex assay setup. (N) Longitudinal evaluation of the optomotor reflex before (pre) and after magnetic microbead injection demonstrates progressive visual acuity decay resulting in significant vision loss by 3 weeks of exposure to OHT (sham: N = 7 mice, OHT: N = 6 mice, two-way ANOVA with Sidak’s multiple comparison post hoc test, ∗∗∗ p < 0.001, n.s.: not significant). c/d, cycles/degree. (O) Quantitative analysis of the optomotor responses at 3 and 4 weeks post-OHT induction shows a significant improvement in visual acuity in mice treated with AAV.SERCA2 relative to AAV.Ctl (sham: N = 13 mice, OHT+AAV.Ctl: N = 6–8 mice, OHT+AAV.SERCA2: N = 7 mice, one-way ANOVA with Tukey’s multiple comparison post hoc test, ∗∗∗ p < 0.001). Data are presented as mean values ± SEM. The cartoon in this figure was generated with BioRender ( https://biorender.com ).

Article Snippet: Paraffin retinal sections were heated for 20 min in citrate buffer (80°C–90°C) followed by incubation in blocking solution (10% normal donkey serum, 1% bovine serum albumin, 0.5% Triton X-100) for 1 h. Retinal sections were incubated with primary antibodies: SERCA2 (4 μg/mL, Invitrogen), Calreticulin (1:250, Invitrogen), and RBPMS (0.25 μg/mL, PhosphoSolutions), followed by anti-mouse Alexa 647, anti-rabbit Alexa Fluor 594, or anti-guinea pig Alexa Fluor 488 (2–4 μg/mL, Molecular Probes).

Techniques: Flow Cytometry, Expressing, Two Tailed Test, Control, Labeling, Injection, Comparison, Generated

Journal: Cell Reports Medicine

Article Title: Endoplasmic reticulum stress-related deficits in calcium clearance promote neuronal dysfunction that is prevented by SERCA2 gene augmentation

doi: 10.1016/j.xcrm.2024.101839

Figure Lengend Snippet:

Article Snippet: Paraffin retinal sections were heated for 20 min in citrate buffer (80°C–90°C) followed by incubation in blocking solution (10% normal donkey serum, 1% bovine serum albumin, 0.5% Triton X-100) for 1 h. Retinal sections were incubated with primary antibodies: SERCA2 (4 μg/mL, Invitrogen), Calreticulin (1:250, Invitrogen), and RBPMS (0.25 μg/mL, PhosphoSolutions), followed by anti-mouse Alexa 647, anti-rabbit Alexa Fluor 594, or anti-guinea pig Alexa Fluor 488 (2–4 μg/mL, Molecular Probes).

Techniques: Virus, Plasmid Preparation, Recombinant, Software

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