Paraffin sections (4 μm) were dried, deparaffinized, and stained with hematoxylin eosin safranin (HES) (Dako). For immunohistochemical staining, 4 µm paraffin sections were incubated with antigen retrieval citrate-based solution pH = 9 (Vector Laboratories) at 95 °C for 15 min. The sections were incubated in Bloxall blocking solution (Vector Laboratories) for 10 min to inactivate endogenous peroxidase. FC receptor blocking reagent (Innovex Biosciences) was then added for 45 min. All sections were incubated in PBS-10% FCS for 30 min with either anti-lamin A/C antibody (dilution 1/200, clone EPR4100, Abcam) or anti-osterix/SP7 antibody (dilution 1/100, Abcam) diluted in PBS-5% FCS-1.5% BSA overnight incubation at 4 °C. The sections were washed with PBS-0.1% Triton and incubated in ImPRESS Reagent anti-rabbit IgG (Vector Laboratories) for 30 min. Then, the sections were washed with PBS-Triton 0.1% and incubated in HistoGreen substrate solution (Linaris) for 1 min. Counter coloration was performed using Fast Nuclear Red (Vector Laboratories). All sections were analyzed using a Pannoramic Midi II slide scanner and Case Viewer software (3D HISTECH, Ltd.).
Anti lamin a c antibody
Manufactured by Abcam
The Anti-lamin A/C antibody is a laboratory reagent used to detect the presence and distribution of lamin A/C proteins in biological samples. Lamin A/C are structural proteins that are part of the nuclear lamina and play a role in regulating gene expression and maintaining nuclear integrity. This antibody can be used in various analytical techniques, such as Western blotting, immunohistochemistry, and immunocytochemistry, to study the expression and localization of lamin A/C in cells and tissues.
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Lab products found in correlation
Maxima first strand cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Caseviewer software, by 3DHISTECH (1 mentions)
Bloxall blocking solution, by Vector Laboratories (1 mentions)
Impress reagent anti rabbit igg, by Vector Laboratories (1 mentions)
Pannoramic midi 2 slide scanner, by 3DHISTECH (1 mentions)
Alexa fluor 488 phalloidin, by Thermo Fisher Scientific (1 mentions)
Paraformaldehyde (pfa), by Electron Microscopy Sciences (1 mentions)
Hoechst 33342, by Thermo Fisher Scientific (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Anti p eif2α antibody, by Abcam (1 mentions)
Anti hmgb1 antibody, by Abcam (1 mentions)
Anti β actin antibody, by Abcam (1 mentions)
Oltipraz, by Merck Group (1 mentions)
Sulforaphane, by Merck Group (1 mentions)
Dual luciferase firefly renilla assay system, by BPS Biosciences (1 mentions)
Itaq sybr green supermix, by Bio-Rad (1 mentions)
Anti nrf2 antibody, by Proteintech (1 mentions)
Dc protein assay reagent, by Bio-Rad (1 mentions)
Live dead baclight bacterial viability kit, by Thermo Fisher Scientific (1 mentions)
5 protocols using anti lamin a c antibody
1
Immunohistochemical Analysis of Lamin A/C and Osterix
2
Immunofluorescence Staining of Cell Junctions
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Cells were fixed with 4% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA) for 10 minutes and washed with PBS before permeabilizing and blocking with permeabilization buffer containing 0.1% triton X-100 in 1% BSA solution for 45 minutes. To stain for E-cadherin, cells were incubated with rabbit monoclonal antibody for E-cadherin (Abcam, Cambridge, MA) at 1:100 dilution for 1 hour in 1% BSA and then incubated with Goat Anti-Rabbit IgG (H + L) antibody (1:200, Life Technologies) at room temperature for 1 hour. To stain for Lamin A/C, the cells were incubated with anti-Lamin A + C antibody (1:500, Abcam, Cambridge, MA) for 1 hour in permeabilization buffer. Cells were then washed and treated with anti-rabbit secondary antibody IgG (H + L) antibody (1:500, Life Technologies) for 1 hour at room temperature. Cells were then stained for F-actin and nucleus with 1:40 Alexa Fluor 488 phalloidin (Invitrogen) and 1:200 Hoechst 33342 (Life Technologies) for 20 minutes at room temperature respectively. Stained cells were imaged with the 60X/1.40 NA oil immersion objective of the Nikon A1 laser scanning inverted confocal microscope (Nikon, Melville, NY).
3
Subcellular Fractionation and Immunoblotting
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A subcellular fractionation protocol provided by Thermo Scientific (Cat No. 78835; Waltham; MA) was used to isolate cytosolic and nuclear protein from cells. The protein concentrations of the cell lysates were measured using the Pierce BCA Protein Assay Reagent Kit (Cat No. 23225). Conditioned medium was collected from the supernatant by centrifugation for 5 min at 1500 rpm. Ten micrograms of proteins were separated by 15% SDS-PAGE. After electrophoresis, proteins were transferred to a polyvinylidene difluoride membrane (Millipore, Bedford, MA). The membrane was incubated with anti-HMGB1 antibody (Cat No. ab18256; Abcam), anti-HMGN1 antibody (Cat No. ab5212; Abcam), anti-p-eIF2α antibody (Cat No. ab32157; Abcam), anti-lamin A+C antibody (Cat No. ab108595; Abcam), or anti-β-actin antibody (Cat No. ab8227; Abcam) overnight at 4°C, followed by incubation with secondary antibodies for 1 h at room temperature. The Promega Western Blot Detection System (Cat No. W1008; Madison; WI) was used to detect immunoreactive proteins.
4
Evaluating Cellular Stress Responses
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Phorbol 12-myristate 13-acetate (PMA), human interferon gamma (IFNγ), lipopolysaccharides (LPS), dimethyl sulfoxide (DMSO), sulforaphane (SFN), dimethyl fumarate (DMF), Oltipraz (OTZ) were purchased from Sigma (MERCK). Wogonin (WG) and anti-GAPDH antibody were obtained from Millipore (MERCK). Dual Luciferase (Firefly-Renilla) Assay System was purchased from BPS Bioscience. RPMI (Roswell Park Memorial Institute) 1640 culture medium, phosphate-buffered saline (PBS), fetal bovine serum, and human GM-CSF were obtained from Eurobio Scientific. Tryptic soy broth and tryptic soy agar were from Conda laboratories (Dutscher). Lipofectamine® LTX & PLUS™ Reagent, Live/Dead™ BacLight™ bacterial viability kit, CellROX™ Green Oxidative Stress Reagent, Maxima First Strand cDNA synthesis kit, and Trizol were obtained from ThermoFisher scientific. Fluoromount-G™ (EMS) was purchased from Euromedex. DC Protein Assay Reagents and iTaq SYBR green supermix were purchased from Bio-Rad. Anti-Lamin A/C antibody was purchased from Abcam. Anti-Nrf2 antibody was obtained from ProteinTech. IRDye® 680CW goat anti-mouse IgG, and IRDye® 800CW Goat anti-Rabbit IgG secondary antibodies were purchased from LI-COR® Biosciences.
5
In Vitro Lamin A/AR Protein Interaction Assay
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In vitro protein interactions with recombinant proteins were performed as follows. 200 ng purified Myc-tagged lamin A (MW: 74 KDa, OriGene; Cat# TP304970) and 200 ng 6XHis-tagged AR (1-556 aa, 63 kDa, Ray Biotech; Cat# RB-14-0003P) were mixed in 500 μl binding buffer (150 mM NaCl, 20mM Tris-HCl; pH-8, 0.1% NP40, 10% glycerol) and incubated overnight at 4°C on a rotary platform. The next day, 20 μl of protein A magnetic beads pre-adsorbed with 0.5 μg of anti-lamin A/C antibody (Abcam, #ab108595) were added to the protein mixture and incubated for 2 hours at RT. Bead-bound protein complexes were separated from the unbound fraction by magnetic pulldown, beads were washed 3 times in binding buffer and then resuspended in 30 μl of SDS-PAGE loading buffer. As a control for specificity, parallel pull-down reactions were performed with AR protein previously denatured by a short heat treatment (20 min at 85°C), or the protein mixture was incubated in high salt buffer (800 mM NaCl). Immunoprecipitates with anti-lamin A/C antibody were analysed in parallel with 5% unbound supernatants by immunoblotting with anti-lamin A/C (Cell Signaling; Cat# #4777) or anti-His (Cell Signaling; Cat#12698) antibodies.
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