Cells were grown in 24-well plates with 0.170mm glass bottom (In Vitro Scientific). Where indicated, the cells were pre-extracted before fixation with pre-extraction buffer (10 mM PIPES, pH 6.8, 100 mM NaCl, 1.5 mM MgCl2, 300 mM sucrose, 0.5% Triton-X 100, 1mM DTT, 5 μg/ml leupeptin, 2 μg/ml aprotinin, 0.1 mM PMSF) for 20 minutes at 4°C, washed by PBS and then immediately fixed with 4% formaldehyde for 15 minutes at room temperature. Cells were stained with primary antibodies: anti-ubiquitylated conjugateed mouse FK2 antibody (1:500; Enzo, cat. n.: BML-PW8810), anti-VCP (1:500; Abcam; cat. n.: ab11433), anti-NPL4 (1:500; Novus Bio, cat. n.: NBP1-82166), HSP70 (1:100; Enzo, cat. n.: ADI-SPA-830), HSF1 (1:500; Cell Signaling, cat. n.: 4356) anti-ubiquitin lys48-specific (1:500; Merck Millipore, clone Apu2), Sumo2/3 (1:500; Abcam, cat. n.: ab3742), TDP-43 (1:300; Proteintech, cat. n.: 10782-2-AP) and appropriate Alexa Fluor 488 and 568 secondary antibodies (Invitrogen, 1:1000). Cytochrome c was stained by Alexa Fluor 555 conjugated mouse anti-cytochrome c antibody according manufacture’s protocol (BD Pharmingen, cat. n.: 558700).
Anti npl4
Manufactured by Novus Biologicals
Sourced in United States, United Kingdom
Anti-NPL4 is a protein that specifically binds to and inhibits the activity of the NPL4 protein. NPL4 is a component of the p97 complex, which is involved in the extraction of ubiquitinated proteins from the endoplasmic reticulum for proteasomal degradation. Anti-NPL4 can be used to study the role of the p97 complex in cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Anti vcp, by Abcam (2 mentions)
Anti ubiquitylated conjugateed mouse fk2 antibody, by Enzo Life Sciences (2 mentions)
Clone apu2, by Abcam (2 mentions)
Alexa fluor 555 conjugated mouse anti cytochrome c antibody, by BD (2 mentions)
Alexa fluor 488 and 568 secondary antibodies, by Thermo Fisher Scientific (2 mentions)
Mounting medium, by Agilent Technologies (1 mentions)
Anti aif, by Cell Signaling Technology (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Glial fibrillary acidic protein (gfap), by Abcam (1 mentions)
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Hematoxylin eosin, by Merck Group (1 mentions)
Neuronal nuclei (neun), by Abcam (1 mentions)
4 protocols using anti npl4
1
Immunohistochemistry of Cellular Stress Markers
2
Immunofluorescence Staining of Cell Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were incubated in primary antibody (anti-NPL4, 1:1 000; Novus Bio; anti-AIF, 1:1 000; Cell Signaling; anti-phospho-Histone H2A.X (Ser139) 1:500, Merk-Millipore) after treatment and fixation, and stained with the secondary antibody (Jackson Laboratory, Bar Harbor, ME, USA), and DAPI (Invitrogen, Carlsbad, CA, USA). Slides were prepared with mounting medium (Dako, Santa Clara, CA, USA) and imaged with a Zeiss microscope. Signal intensity was quantified with ImageJ (National Institute of Health).
3
Immunohistochemistry of Cellular Stress Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were grown in 24-well plates with 0.170mm glass bottom (In Vitro Scientific). Where indicated, the cells were pre-extracted before fixation with pre-extraction buffer (10 mM PIPES, pH 6.8, 100 mM NaCl, 1.5 mM MgCl2, 300 mM sucrose, 0.5% Triton-X 100, 1mM DTT, 5 μg/ml leupeptin, 2 μg/ml aprotinin, 0.1 mM PMSF) for 20 minutes at 4°C, washed by PBS and then immediately fixed with 4% formaldehyde for 15 minutes at room temperature. Cells were stained with primary antibodies: anti-ubiquitylated conjugateed mouse FK2 antibody (1:500; Enzo, cat. n.: BML-PW8810), anti-VCP (1:500; Abcam; cat. n.: ab11433), anti-NPL4 (1:500; Novus Bio, cat. n.: NBP1-82166), HSP70 (1:100; Enzo, cat. n.: ADI-SPA-830), HSF1 (1:500; Cell Signaling, cat. n.: 4356) anti-ubiquitin lys48-specific (1:500; Merck Millipore, clone Apu2), Sumo2/3 (1:500; Abcam, cat. n.: ab3742), TDP-43 (1:300; Proteintech, cat. n.: 10782-2-AP) and appropriate Alexa Fluor 488 and 568 secondary antibodies (Invitrogen, 1:1000). Cytochrome c was stained by Alexa Fluor 555 conjugated mouse anti-cytochrome c antibody according manufacture’s protocol (BD Pharmingen, cat. n.: 558700).
4
Histopathology Analysis of Brain Tissue
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Brains of symptomatic animals were perfused with 4% paraformaldehyde (Sigma Aldrich) and switched to 10% formalin after 24h for long-term preservation. All samples were paraffin-embedded by the Johns Hopkins University Histopathology Core and stained with hematoxylin-eosin (Sigma Aldrich), and with immunohistochemistry for anti-NPL4 (1:100; Novus Bio), Ki67 (1:100; Cell Signaling), Cleaved-Caspase-3 (1:100; Cell Signaling), GFAP (1:500, Abcam) and NeuN (1:500, Abcam, Cambridge, UK). Brains were evaluated for tumor growth, signs of toxicity, and tissue damage.
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!