Transfection was performed in the same manner as Lamin A/C knockdown, but cells were instead seeded on poly-D-lysine coated glass-bottom dishes at 15,000 cells cm−2. After the 72 hour incubation, samples were rinsed and directly fixed with 10% neutral buffered formalin for 15 minutes, preserving the attached morphology. Cells were then permeabilized with 0.1% Triton-X 100 for 15 minutes; blocked with 2% BSA for 20 minutes; incubated overnight with mouse anti-lamin A/C (ab8984, Abcam), diluted 1:250; and incubated with goat anti-mouse IgG Alexa fluor 594 conjugate (A-11032, ThermoFisher), diluted 1:500, for one hour. All steps were performed at room temperature, except for incubation with primary antibody at 4°C, and samples were rinsed three times with PBS between each step. Antibody solutions were prepared in 1% BSA.
Ab8984
Manufactured by Abcam
Sourced in United Kingdom, United States
Ab8984 is a primary antibody. It is designed for use in immunohistochemistry applications.
Automatically generated - may contain errors
Lab products found in correlation
Ab16048, by Abcam (4 mentions)
Hoechst 33342, by Thermo Fisher Scientific (3 mentions)
Ab49311, by Abcam (2 mentions)
Lsm 880, by Zeiss (2 mentions)
A11032, by Thermo Fisher Scientific (1 mentions)
Ab64159, by Abcam (1 mentions)
Ab15277, by R&D Systems (1 mentions)
Frozen section compound, by Leica Microsystems (1 mentions)
M3559, by Agilent Technologies (1 mentions)
Blocking one, by Abcam (1 mentions)
Mab4470, by R&D Systems (1 mentions)
Phalloidin red 594, by Beyotime (1 mentions)
Alexa fluor 488 goat anti mouse, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 568 goat anti rabbit, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 568 goat anti mouse, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 goat anti rabbit, by Thermo Fisher Scientific (1 mentions)
Paraformaldehyde (pfa), by Solarbio (1 mentions)
Ab9134, by Abcam (1 mentions)
Spectramax m5 plate reader, by Molecular Devices (1 mentions)
Ab179466, by Abcam (1 mentions)
14 protocols using ab8984
1
Formalin-fixed Lamin A/C Immunostaining
2
Immunohistochemical Analysis of Skeletal Muscle
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Cells and skeletal muscle tissues were fixed with 4% paraformaldehyde in PBS for 15 min at 4°C, prior to embedding in Frozen Section Compound (Leica Microsystems) for cryosections. Fixed samples were incubated with 0.1%Triton X-100 in PBS for 5 min, BlockingOne (Nakacai Tesque) for 30 min, and anti-GFP (Molecular Probes A6455; diluted 1:500), anti-PAX3 (DSHB AB528426; diluted 1:100), anti-PAX7 (DSHB AB528428; diluted 1:100), anti-myogenin (DAKO M3559; diluted 1:100), anti-MyoD (Abcam ab64159; diluted 1:500), anti-laminin (Enzo Life Sciences ALX-804-190; diluted 1:500), anti-dystrophin (Abcam ab15277; diluted 1:200), anti-MyHC (R&D MAB4470; MF20, diluted 1:200), anti-FSP1 (Abcam ab124805; diluted 1:200), anti-lamin A/C (Abcam ab8984; diluted 1:200), and anti-Ki67 (Abcam ab15580; diluted 1:500) antibodies in 5% BlockingOne overnight at 4°C. After three washes with 0.1% Tween 20 in PBS, cells were incubated with Alexa-conjugated anti-mouse immunoglobulin G1 (IgG1), mouse IgG2b, rabbit IgG, or rat IgG antibodies (Molecular Probes; diluted 1:500). Cells were washed and mounted in ProLong Diamond antifade reagent with DAPI (Molecular Probes). Images were collected and processed by BZ-X710 microscopy.
3
Immunofluorescence Staining of Cellular Structures
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Followed by washing with PBS three times, cells were fixed with 4% paraformaldehyde (Solarbio, Beijing, China) for 10 min at room temperature. PBS was used to wash these cells after fixation, and 0.1% Triton X-100 was used for 5 min. Then, blocking was performed by 5% bovine albumin serum for 30 min at room temperature. Cells were incubated overnight at 4 °C with primary antibodies (mouse anti-Lamin A/C (1:500, ab8984, Abcam, London, UK), Rabbit anti-MKL1 (1:500, ab49311, Abcam), rabbit anti-β-catenin (1:500, D10A8, Cell Signaling Technology, Danvers, MA, USA), and Phalloidin Red-594 (1:500, Beyotime, C2203S, Shanghai, China)). Cells were again washed three times, and the second antibodies (AlexaFluor488 goat anti-mouse (1:500), AlexaFluor488 goat anti-rabbit (1:500), AlexaFluor568 goat anti-mouse (1:500), AlexaFluor568 goat anti-rabbit (1:500), (Invitrogen, USA)) were added. Cells were incubated for 1 h in the dark. These cells were imaged under confocal microscopy, and the data were analyzed using the SPSS® Statistics 20 software.
4
Quantifying SUFU Variant Effects
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peGFP-C1 SUFU constructs were nucleofected into ASZs and plated at 50% confluency into a 96-well plate in 6 biological replicates. After 24 hours, cells were treated with 154CF (Life Technologies) supplemented with Real Time Glow reagent per manufacture’s instructions (Promega). Time points were taken every few hours for 2 days to generate growth curves for each SUFU variant. Each well was normalized to itself after a three-hour equilibration to control for plating variation. Luminescence was measured by the SpectraMax M5 plate reader (Molecular Devices) at 37°C over a 1.5 second interval. SUFU overexpression via nucleofection in ASZ001 cells was confirmed via immunoblotting. Primary antibodies against HA (Abcam ab9134) and Lamin A + C (Abcam ab8984) as a loading control were used.
5
Immunofluorescent Analysis of Cellular Organelles
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Isolated lung tissues were fixed in 4% paraformaldehyde, embedded in paraffin, cut into 4‐μm‐thick sections, and stained with haematoxylin and eosin. Infiltrating neutrophils were revealed by immunofluorescence using anti‐mouse Ly‐6G/Ly‐6C (Gr‐1) antibody (BioLegend) followed by Alexa 488‐conjugated‐goat anti‐rabbit IgG (Abcam) secondary antibody.
hMSCs were fixed with 4% paraformaldehyde for 15 min, then washed with PBS and permeated with 0.5% Triton X‐100 (V900502‐100ML, Sigma) in PBS for 15 min. The nuclei were stained with Hoechst 33324 (H3570, Thermo Fisher Scientific). The antibodies against Vimentin (ab92547, abcam), HP1α (ab109028, abcam), Lamin B1 (ab16048, abcam), Lamin A/C (ab8984, abcam), H3K9Me2/3(5327, CST), SP100 (ab167605, abcam), and PML (ab179466 and ab96051, abcam) were used as primary antibodies. Secondary antibodies were Alexa 488‐conjugated‐goat anti‐rabbit IgG (Abcam) and Alexa 555‐conjugated‐goat antimouse IgG (Thermo Fisher Scientific). Images were taken by a laser‐scanning confocal microscope (Leica TCS SP8, Leica, Germany).
hMSCs were fixed with 4% paraformaldehyde for 15 min, then washed with PBS and permeated with 0.5% Triton X‐100 (V900502‐100ML, Sigma) in PBS for 15 min. The nuclei were stained with Hoechst 33324 (H3570, Thermo Fisher Scientific). The antibodies against Vimentin (ab92547, abcam), HP1α (ab109028, abcam), Lamin B1 (ab16048, abcam), Lamin A/C (ab8984, abcam), H3K9Me2/3(5327, CST), SP100 (ab167605, abcam), and PML (ab179466 and ab96051, abcam) were used as primary antibodies. Secondary antibodies were Alexa 488‐conjugated‐goat anti‐rabbit IgG (Abcam) and Alexa 555‐conjugated‐goat antimouse IgG (Thermo Fisher Scientific). Images were taken by a laser‐scanning confocal microscope (Leica TCS SP8, Leica, Germany).
6
Western Blot Analysis of Cellular Proteins
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Samples were electrophoresed through 10% SDS-PAGE, followed by blotting of the proteins onto nitrocellulose membranes. After blocking with skim milk, the blots were incubated overnight with the antibodies listed below, washed, and incubated with the appropriate horseradish peroxidase (HRP)-conjugated secondary antibody. An antibody against tubulin (T5168) was purchased from Sigma-Aldrich. Antibodies against IGF1R (#3027), insulin receptor (IR) β-subunit (#3025), SP1 (#5931) and SUMO-1 (#2A12) were obtained from Cell Signaling Technology Inc (Beverly, MA, USA). Antibodies against lamin A/C (ab8984) and lamin B1 (ab16048) were purchased from Abcam (Cambridge, MA, USA). The secondary antibodies were goat anti-rabbit IgG (1:50,000) and donkey anti-mouse IgG (1:25,000; Jackson ImmunoResearch Laboratories, West Grove, PA, USA). Proteins were detected using the SuperSignal West Pico Chemiluminescent Substrate (Pierce, Waltham, MA, USA).
7
Subcellular Protein Fractionation and Western Blot
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Whole-cell lysates or nuclear protein was extracted using a protein extraction buffer (Beyotime, Shanghai, China) or nucleoprotein extraction kit (Sangon Biotech, C500009), respectively. Proteins were resolved by SDS-PAGE and transferred onto nitrocellulose (NC) membranes using standard methods. Primary antibodies used as follows: MPC1 (ab74871, Abcam), β-catenin (ab32572, Abcam), lamin A/C (ab8984, Abcam), and GAPDH (ab9485, Abcam). Species-specific secondary antibodies used as follows: IRDye 680 Goat anti-Mouse IgG (LI-COR) and IRDye 800 Goat anti-rabbit IgG (LI-COR).
8
Immunoblotting of NF-κB and Related Proteins
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The cell lysates were fractioned by 10% SDS-PAGE and then blotted onto nitrocellulose membranes (Bio-Rad, Hercules, CA, USA). The membranes were incubated overnight with primary antibodies at a dilution of 1:1000, with the exception of anti-β-tubulin (1:2000). The antibodies used for immunoblotting were: rabbit anti-NF-κB p50 (sc-7178, Santa Cruz), rabbit NF-κB p65 (sc-7151, Santa Cruz), rabbit anti-Bcl-3 (sc-185, Santa Cruz), rabbit anti-STAT3 (sc-482, Santa Cruz), rabbit anti-IRF2 (ab124744, Abcam), rabbit anti-IκBα (9242S, Cell Signaling Technology, Danvers, MA, USA), mouse anti-phospho-IκBα (5A5, Cell Signaling), mouse anti-β-tubulin (T8328, Sigma-Aldrich, St. Louis, MO, USA), and mouse anti-lamin A+C (ab8984, Abcam). The membranes were then incubated with IRDye700-labeled donkey anti-mouse or anti-rabbit, or IRDye800-labeled donkey anti-mouse (LI-COR Biosciences) at a 1:5000 dilution. The blots were detected using an Odyssey® Infrared Imaging System (LI-COR Biosciences).
9
Validating Nuclear and Cytoplasmic Fractions
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Western blots to determine the purity of nuclear and cytoplasmic fractions from TurboNuclease or DNase-treated T47D or Flag-AGO2 cells were performed as before (Gagnon et al. 2014b (link)). For comparing nuclear and cytoplasmic fractions by Western blot, the same cell equivalents of extract were separated by electrophoresis (one-half the volume of nuclear extract per one volume of cytoplasmic). Blocked Western blot membranes (Hybond-C Extra, GE Healthcare Life Sciences) were incubated with the following primary antibodies for 16 h at 4°C in PBST (PBS + 0.05% TWEEN-20) + 5% milk with rocking: anti-AGO2 at 1:1000 (Abcam, ab57113), anti-Calreticulin at 1:1000 (Cell Signalling, 2891S), anti-Histone H3 at 1:10,000 (Abcam, ab1791), anti-Lamin A/C at 1:1500 (Abcam, ab8984), anti-GAPDH at 1:600 (Abcam, ab9484), anti-RNA polymerase II at 1:4000 (Millipore, 05-623).
10
Immunofluorescence Staining of Bone Marrow-Derived Macrophages
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BMDMs were rinsed twice with 1X PBS, followed by fixation using 4% paraformaldehyde (18814–20, Polysciences Inc.) in 1X PBS for 20 min. BMDMs were washed and permeabilized with 0.3% Triton X-100 (Sigma) in 1X PBS for 10 min. After washing twice, BMDMs were treated with 2% BSA (blocking solution) for 60 min before incubating with primary antibody (diluted in 2% BSA) at 4°C overnight. Primary antibodies against Lamin-A/C (1:100, ab8980 Abcam; 1:100, ab8984 Abcam; 1:100 4777S Cell Signalling), phospho-Ser22-Lamin-A/C (pSer22) (1:100, 2026 Cell Signalling) and p65 (NFκB p65 1:300, sc-8008 Santa Cruz) were used for staining. BMDMs were then washed with blocking solution and incubated with the corresponding secondary antibodies, diluted in blocking solution, along with the nuclear stain Hoechst-33342 (1:1000, 62249 Life Technologies) for 45 min. Filamentous actin (F-actin) was labelled using phalloidin Alexa-Fluor 568 (1:200, Life Technologies). All antibodies used in this study have previously been extensively used and validated for immunofluorescence studies.
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