To measure apoptotic activity of cardiomyocytes indicated by DNA stand breaks, terminal deoxynucleotidyl transferase‐mediated dUTP nick end labeling (TUNEL; In Situ Cell Death Detection FITC Kit or TMR red, Roche) assays were performed on cardiomyocytes or heart tissues, which were immunostained with anti‐α‐actinin antibody (Sigma‐Aldrich). Cells planted on cover slips or cryosections of mouse heart were fixed in 4% paraformaldehyde for 1 hour, blocked with methanol with 3% H2O2 for 10 minutes, and permeabilized with 0.1% Triton X–100 in 0.1% sodium citrate for 2 minutes. Cryosections were incubated with anti‐α‐actinin antibodies (1:200 dilution; Sigma‐Aldrich) in a humidified chamber for 1 hour, followed by TUNEL staining for 1 hour and 4’,6‐diamidino‐2‐phenylindole staining for nuclei for 5 minutes. Laser scanning confocal microscopy (FV300, Olympus) was used to detect TUNEL‐positive cells in randomly selected fields.
Anti α actinin antibody
Manufactured by Merck Group
Sourced in Germany, United States
The Anti-α-actinin antibody is a laboratory reagent used for the detection and analysis of the α-actinin protein. α-actinin is a key structural component of the actin cytoskeleton, which plays a crucial role in cellular organization and function. This antibody can be utilized in various immunoassay techniques, such as Western blotting and immunohistochemistry, to identify and quantify the presence of α-actinin in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Fv300, by Olympus (3 mentions)
In situ cell death detection fitc kit, by Roche (2 mentions)
Tmr red, by Roche (2 mentions)
Bz 9000 microscope, by Keyence (1 mentions)
Ecl advance chemiluminescence kit, by GE Healthcare (1 mentions)
Phenylephrine, by Merck Group (1 mentions)
Anti p70s6k, by Cell Signaling Technology (1 mentions)
Anti phospho p70s6k, by Cell Signaling Technology (1 mentions)
Anti p110α, by Cell Signaling Technology (1 mentions)
Anti phospho mtor, by Cell Signaling Technology (1 mentions)
Anti eef2, by Cell Signaling Technology (1 mentions)
Anti akt, by Cell Signaling Technology (1 mentions)
Angii angiotensin 2, by Merck Group (1 mentions)
Anti mtor, by Cell Signaling Technology (1 mentions)
Anti phospho akt, by Cell Signaling Technology (1 mentions)
Anti phospho erk1 2, by Cell Signaling Technology (1 mentions)
Anti vimentin, by Cell Signaling Technology (1 mentions)
Anti erk1 2, by Cell Signaling Technology (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Gel logic 2200 pro imaging system, by Carestream (1 mentions)
23 protocols using anti α actinin antibody
1
Quantifying Cardiomyocyte Apoptosis via TUNEL
2
Immunofluorescence Staining and Imaging of Tissue Cryosections
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cryosections of the border region (4 μm) were fixed in 4% paraformaldehyde for 2 hours, permeabilized with 0.1% Triton X–100 in 0.1% sodium citrate for 10 minutes, and blocked with 5% bovine serum albumin in PBS with Tween 20 for 1 hour. Cryosections were incubated with anti‐α‐actinin antibody (1:500; Sigma‐Aldrich) and anti‐TGFβR3 antibody (1:100; Sigma‐Aldrich) in a humidified chamber overnight at 4°C, followed by 4’,6‐diamidino‐2‐phenylindole staining for nuclei for 5 minutes. Laser scanning confocal microscopy (FV300, Olympus) was used to image slides in randomly selected fields.
3
Culturing Cardiomyocytes with ACF Conditioned Media
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
NRCM were cultured in cardiomyocyte maintenance medium (68% DMEM, 17% M199, 10% horse serum, 5% FBS, 2.5 μg ml−1 amphotericin B and 1 μM BrdU), whereas adult rat cardiomyocytes were cultured in pre-equilibrated myocyte culture medium (Eagle's MEM w/HBSS containing; 1 mg ml−1 BSA, 10 mM BDM, 100 U ml−1 penicillin-G, 2 mM Glutamine, 5 μg ml−1 insulin, 5 μg ml−1 transferring and 5 ng ml−1 selenium) for 24 h. Next, the medium was replaced with conditioned medium from ACFs culture. To prepare the conditioned medium, ACFs isolated from WT or PMCA4−/− mice were plated in cardiomyocyte maintenance medium for 24 h. The media were collected and used to culture NRCM. Cells were then stimulated with phenylephrine (30 μM) for 72 h with or without 0.2 μg ml−1 anti-sFRP2 antibody (Abcam) for 72 h. To specifically visualize cardiomyocytes, cells were stained with anti-α-actinin antibody (Sigma). The cell size was then measured using ImageJ software (NIH). The level of sFRP2 in the conditioned medium of WT or PMCA4−/− ACFs was determined using ELISA (My BioSource) following the m manufacturer's protocol.
4
Cardiomyocyte Immunofluorescence and Morphometry
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immunofluorescence staining and cell surface area measurements were performed as detailed earlier [34 (link)]. Briefly, cardiomyocytes cultured on coverslips in 12x well culture plates, either transduced with Cfl2 overexpression/knockdown adenoviral particles, or transfected with miR-301a mimic/inhibitor, were washed 2x with PBS and fixed with 4% paraformaldehyde for 10 min. Fixed cells were washed 2x with PBS followed by a common step of permeabilization and blocking with 0.1% Triton X-100 in 2.5% BSA for 1 h at room temperature. Cells were then incubated for 1 h with primary anti–α-actinin antibody (1:200; Sigma-Aldrich), 5x washes with PBS, followed by the incubation with respective secondary antibody conjugated to Cy3 (Dianova) and DAPI for nuclear staining. After washings with PBS, coverslips were mounted on glass-slides using Fluoromount (Biozol). Immunofluorescence images were captured using BZ-9000 microscope (Keyence). Cell surface area was measured using HybridCellCount module BZ-II Analyzer software (Keyence).
5
Quantification of PKC δ Phosphorylation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
20 µg of protein extracted in the presence of protease and phosphatase inhibitors was separated on a 12% precast SDS gel (Thermo Scientific). This was followed by a protein transfer to a PVDF membrane, blocking (5% BSA in 1xTBS, 0.01% Tween-20) and an overnight incubation at 4°C in primary antibody (1∶1000) against Thr505 phosphorylated PKC δ (Cell Signaling Technologies). HRP-conjugated secondary anti- rabbit IgG antibody (Promega) was incubated at 1∶20,000 for 1 hr at room temperature prior to visualization with the ECL advance chemiluminescence kit (GE Healthcare). As a positive control, MCF-7 breast cancer cells were treated with 0.2 µg/ml Dox for 4 hours in the absence of foetal bovine serum (FBS).
Quantification - membranes were stripped and re-probed using an anti α-actinin antibody (Sigma Aldrich) as previously described [30] (link). Densitometry was performed using a FluoChem 8800 (Alpha Innotech Corporation) and normalised to α actinin.
Quantification - membranes were stripped and re-probed using an anti α-actinin antibody (Sigma Aldrich) as previously described [30] (link). Densitometry was performed using a FluoChem 8800 (Alpha Innotech Corporation) and normalised to α actinin.
6
Cardiomyocyte Hypertrophy Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
We analysed cardiomyocyte cell circumference and measured expression of the hypertrophic marker, brain natriuretic peptide (BNP), using a BNP‐luciferase reporter in cardiomyocytes. The day before the experiments, NRCM were cultured on laminin‐coated coverslips in a maintenance medium with 1% serum. They were then treated with phenylephrine (Sigma) at a dose of 50 μM for 72 hr with or without the addition of XMU‐MP‐1 at 1–5 μM. To determine cardiomyocyte size, cells were stained with an anti‐α‐actinin antibody (Sigma). The cell size (cell circumference) was then measured using ImageJ software. These experiments were carried out using NRCM isolated from six separate litters of rat neonates, to give N = 6 independent biological replications. At least n = 50 cardiomyocytes were analysed per treatment group in each experiment. To minimise variability due to different conditions between independent experiments, we normalised all cell size values against the mean value of the control group. Data are presented as fold induction relative to the mean value of the control group.
7
Antibody-based Signaling Pathway Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The anti-α-actinin antibody (A7811) was from Sigma-Aldrich. The anti-phospho AKT (#9271), anti-AKT (#9272), anti-phospho mTOR (#2971), anti-mTOR (#2972), anti-phospho p70s6k (#9206), anti-p70s6k (#9202), anti-phospho eEF2 (#2331), anti-eEF2 (#2332), anti—phospho ERK1/2 (#9101), anti-ERK1/2 (#9102), anti-Vimentin (#5741) and anti-p110α (#4249) antibodies were from Cell Signaling Technology. The anti- α-Tubulin (sc-5286), anti-p110α (sc-7174) and anti P110γ (sc-7177) antibody were from Santa Cruz Biotechnology. The anti-Pik3ip1 (16826-1-AP) antibody was from proteintech. AngII (Angiotensin II) was purchased from Sigma-Aldrich, LY294002 was from Calbiochem and Human IGF-1 (insulin-like growth factor-1) was from R&D Systems.
8
Western Blot Analysis of α-Actinin
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were lysed in lysis buffer according to previous report [24 (link)] and were centrifuged at 12,000 g for 20 min, at 4°C. For each sample, 20 μg total protein was electrophoresed through a 10% (w/v) acrylamide gel and then transferred to a nitrocellulose membrane (Bio-Rad Laboratories, Hercules, CA, USA). The blots were incubated at 4°C overnight with anti-α-actinin antibody (Sigma-Aldrich), and the resulting bands were detected by using diaminobenzidine- (DAB-) based horseradish peroxidase (HRP) reaction. Intensities of the bands were semiquantified using a Gel Logic 2200 PRO imaging system (Carestream Molecular Imaging, New Haven, Connecticut, USA).
9
Nur77 Knockdown in Cardiac Myocytes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
NRVMs were transfected with a final concentration of 175 nM Nur77 siRNA (SMARTpool, Dharmacon, Lafayette, CO, USA) or a non-targeting control siRNA for 6 h using Lipofectamine RNAiMAX (Invitrogen) according to manufacturer’s instructions. For assessment of Nur77 knockdown, NRVMs were cultured for 48 h after transfection and were subsequently stimulated with 25 µm isoproterenol or equivalent volume of PBS for 1 h. For cell size experiments, NRVMs were stimulated with 25 µm isoproterenol or PBS for 48 h after transfection. Cells were fixed with 4% paraformaldehyde (Roth, Karlsruhe, Germany) and stained with anti-α-Actinin antibody (Sigma Aldrich), Hoechst and anti-mouse AlexaFluor488-conjugated secondary antibody (Molecular Probes, Eugene, OR, USA). Size was measured in ≥250 Actinin-positive cells per group using ImageJ software (U.S. National Institutes of Health, Bethesda, Maryland, USA).
10
miR-185 Modulation of Cardiomyocyte Hypertrophy
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
NRVMs were grown on 1% gelatin coated-glass coverslips (18-mm diameter). The cells then were transfected with miRNA mimic or hairpin inhibitor for miR-185, or for the negative control, with DharmaFECT-3 reagent. Twenty four hours after transfection, cells were stimulated with 10 nmol/L ET-1 for another 48 h to induce cardiac hypertrophy. Next, cells were fixed with 4% paraformaldehyde for 15 min at RT, washed 3 times with PBS, permeabilized with 0.1% Triton X-100, and blocked with 10% bovine serum albumin (Sigma-Aldrich) in PBS for 30 min. The cells then were incubated with anti-α-actinin antibody (Sigma-Aldrich) in blocking buffer O/N at 4°C, rinsed six times with PBS, incubated with secondary antibody (Alexa 594-conjugated anti-mouse IgG antibody, Molecular Probe) in the blocking buffer for 2 h at 37°C, rinsed three times with PBS, and mounted. The slides were examined with an LSM 700 confocal laser scanning microscope (Carl Zeiss).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!