Overview

  • Product name
    Anti-Nestin antibody [Rat 401]
    See all Nestin primary antibodies
  • Description
    Mouse monoclonal [Rat 401] to Nestin
  • Host species
    Mouse
  • Specificity
    The clone number has been updated from (2Q178) to (Rat 401) both clone numbers name the same antibody clone.
  • Tested applications
    Suitable for: ICC/IF, IHC-FoFr, IHC-P, IHC-Fr, WBmore details
  • Species reactivity
    Reacts with: Mouse, Rat
    Does not react with: Sheep, Cat, Human, Monkey
  • Immunogen

    Full length native protein (purified) corresponding to Rat Nestin.

  • Positive control
    • WB: Mouse brain and rat brain whole tissue lysates. IHC-P: Rat Brain 6 weeks (cerebellum sagittal, coronal rest) tissue sections. ICC/IF: Mouse embryonic stem cells.
  • General notes

    This antibody clone is manufactured by Abcam. If you require a different buffer formulation or a particular conjugate for your experiments, please contact orders@abcam.com.

Properties

Applications

Our Abpromise guarantee covers the use of ab6142 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
ICC/IF 1/20 - 1/200. Cells fixed with 4% paraformaldehyde buffered with 50mM sodium borate at pH 9.5.
IHC-FoFr Use at an assay dependent concentration. PubMed: 18562299
IHC-P Use a concentration of 0.1 µg/ml. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

Tissue fixed with 4% paraformaldehyde at pH 7.4 for light microscopy.

IHC-Fr Use a concentration of 0.1 - 1 µg/ml. Tissue fixed with 4% paraformaldehyde at pH 7.4 for light microscopy.
WB Use a concentration of 1 µg/ml. Predicted molecular weight: 200 kDa.

Block with milk or BSA but do not dilute primary antibody in buffer containing milk.

Target

  • Function
    Required for brain and eye development. Promotes the disassembly of phosphorylated vimentin intermediate filaments (IF) during mitosis and may play a role in the trafficking and distribution of IF proteins and other cellular factors to daughter cells during progenitor cell division. Required for survival, renewal and mitogen-stimulated proliferation of neural progenitor cells.
  • Tissue specificity
    CNS stem cells.
  • Sequence similarities
    Belongs to the intermediate filament family.
  • Developmental stage
    Upon terminal neural differentiation, nestin is down-regulated and replaced by neurofilaments.
  • Post-translational
    modifications
    Constitutively phosphorylated. This increases during mitosis when the cytoplasmic intermediate filament network is reorganized.
  • Information by UniProt
  • Database links
  • Alternative names
    • ESTM 46 antibody
    • FLJ 21841 antibody
    • FLJ21841 antibody
    • Intermediate filament protein antibody
    • Nbla00170 antibody
    • nes antibody
    • NEST_HUMAN antibody
    • Nestin antibody
    see all

Images

  • All lanes : Anti-Nestin antibody [Rat 401] (ab6142) at 1 µg/ml

    Lane 1 : Mouse E12 brain tissue lysate
    Lane 2 : Mouse E14 brain tissue lysate
    Lane 3 : Mouse E16 brain tissue lysate
    Lane 4 : Mouse E18 brain tissue lysate
    Lane 5 : Rat E12 brain tissue lysate
    Lane 6 : Rat E14 brain tissue lysate
    Lane 7 : Rat E16 brain tissue lysate
    Lane 8 : Rat E18 brain tissue lysate

    Lysates/proteins at 20 µg per lane.

    Performed under reducing conditions.

    Predicted band size: 200 kDa



    This blot was produced using a 3-8% Tris-Acetate gel under the Tris-Acetate buffer system. The gel was run at 150V for 60 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 3% milk before being incubated with ab6142 overnight at 4°C. Antibody binding was detected using Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) at 1/20000 dilution for 1 hour at room temperature before imaging.

  • IHC image of Nestin staining in a section of formalin-fixed paraffin-embedded normal rat brain performed on a Leica BONDTM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20mins. The section was then incubated with ab6142, 0.05ug/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

    For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

  • Mesencephalic neural stem cells were washed three times in PBS, fixed in 4% paraformaldehyde for 10 min at room temperature (RT), and permeabilized with PBS plus 0.25% Triton X-100 (PBST) for 10 min at RT (or 15 min at RT if the target antigen was a nucleoprotein). After three washes in PBS, the cells were blocked with 1% BSA for 30 min at RT and incubated with ab6142 staining Nestin (Red) at 1/1000 dilution.

    Immunolabeling was visualized by incubation (1 h at RT) with rhodamine-conjugated Affinipure goat anti-mouse or anti-rabbit IgG (1∶100). After three washes in PBS and two in deionized water, immunolabeled cells were counterstained with 50 µl DAPI at 37°C for 10 min and washed in sequence with methanol and ethanol. Cells were observed under a fluorescence microscope.

    Fluorescence photomicrographs show that all neurospheres were immunoreactive for the NSCs marker nestin. The neurospheres of peGFPN1-GDNF-transfected mNSCs (termed GDNF-mNSCs) were strong immunopositive for GDNF compare to mNSCs transfected with peGFPN1. Green: FITC, Red: Nestin. Scale bar = 50 µm.

  • ab6142 at 1/500 dilution staining Nestin in rat brain tissue (green) by immunohistochemistry (frozen sections). Sections were methanol fixed, permeabilized in 0.1% Triton X-100 prior to blocking in 2.5% BSA for 16 hours at 4°C and then incubated with ab6142, for 1 hour at 37°C. Alexa fluor® 680 goat polyclonal to mouse Ig, diluted 1/1000 was used as the secondary antibody.

    See Abreview

  • ab6142 staining mouse embryonic stem cells by immunocytochemistry/ immunofluorescence. Cells were PFA fixed and permeabilized in 0.4% Triton X-100 prior to blocking in 5% serum for 1 hour at 25°C. The primary antibody was diluted 1/500 and incubated with the sample for 20 hours at 4°C. Alexa fluor® 568 goat polyclonal to mouse Ig, diluted 1/400, was used as the secondary antibody.

    See Abreview

  • ab6142 staining Nestin in mouse brain tissue sections by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with acetone, permeabilized with Triton X-100 in PBS and blocked with 5% BSA for 60 minutes at room temperature. Samples were incubated with primary antibody (1/1000 in PBST) for 12 hours at 4°C. An Alexa Fluor® 488-conjugated goat anti-mouse IgG monoclonal was used as the secondary antibody at a dilution of 1/200.

    See Abreview

  • ab6142 at 1/200 dilution staining Nestin in mouse Cor1 neural stem cells by Immunocytochemistry/ Immunofluorescence. Cells were fixed using 4%PFA for 15mins at RT. After x3 PBS rinses over 15mins, cells were blocked/permeabilised using primary Antibody dilution buffer for 15mins befpre application of primary Ab diluted in same buffer. Secondary conjugate was diluted in same buffer, with addition of 1/10K Hoechst, to stain nuclei. After 60mins cells were rinsed x3 over 15mins before coverslipping using Mowiol containing DABCO and azide.

    See Abreview

  • ab6142 staining adult mouse brain tissue section by Immunohistochemistry (Formalin/PFA-fixed, paraffin embedded sections). Tissue underwent fixation in paraformaldehyde, heat mediated antigen retrieval in Sodium Citrate, permeabilization in 1% Triton buffer and blocking in 10% serum for 1 hour at 25°C. The primary antibody, diluted 1/200 (PBS, 2% Donkey serum, 0.2% Triton) for 16 hours at 4°C.  An Alexa Fluor® 488 conjugated donkey polyclonal to mouse Ig, diluted 1/500 was used as the secondary.

    See Abreview

References

This product has been referenced in:
  • Gholinejad M  et al. Adenosine decreases oxidative stress and protects H2O2-treated neural stem cells against apoptosis through decreasing Mst1 expression. Biomed Rep 8:439-446 (2018). Read more (PubMed: 29732147) »
  • Lin GQ  et al. Transplanted human neural precursor cells integrate into the host neural circuit and ameliorate neurological deficits in a mouse model of traumatic brain injury. Neurosci Lett 674:11-17 (2018). IHC . Read more (PubMed: 29501684) »
See all 95 Publications for this product

Customer reviews and Q&As

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1-10 of 24 Abreviews

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunohistochemistry (Frozen sections)
Sample
Rat Tissue sections (Brain)
Permeabilization
Yes - Triton-X 100
Specification
Brain
Blocking step
Donkey serum as blocking agent for 10 minute(s) · Concentration: 2% · Temperature: 23°C
Fixative
Paraformaldehyde

Alyssa Cortella

Verified customer

Submitted Aug 24 2018

Application
Immunohistochemistry (PFA perfusion fixed frozen sections)
Sample
Mouse Tissue sections (Brain)
Antigen retrieval step
None
Permeabilization
Yes - TritonX-100
Specification
Brain
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 4°C
Fixative
Paraformaldehyde

Kyungjoo Seong

Verified customer

Submitted Nov 02 2016

Application
Immunocytochemistry
Sample
Mouse Cell (Mouse neural stem cell)
Permeabilization
Yes - tritonx100
Specification
Mouse neural stem cell
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 4°C
Fixative
Paraformaldehyde

Kyungjoo Seong

Verified customer

Submitted Nov 02 2016

Application
Immunohistochemistry (Frozen sections)
Sample
Mouse Tissue sections (brain)
Permeabilization
Yes - Triton
Specification
brain
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: RT°C
Fixative
Acetone

Abcam user community

Verified customer

Submitted Feb 09 2016

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunohistochemistry (Frozen sections)
Sample
Rat Tissue sections (adult rat heart tissue)
Permeabilization
Yes - 0.1% Triton
Specification
adult rat heart tissue
Blocking step
Serum as blocking agent for 20 minute(s) · Concentration: 2% · Temperature: 20°C
Fixative
Paraformaldehyde

Abcam user community

Verified customer

Submitted Jan 28 2016

Application
Western blot
Loading amount
400000 cells
Gel Running Conditions
Reduced Denaturing (12%)
Sample
Mouse Cell lysate - whole cell (Mouse Neuroblastoma (Neuro2A))
Specification
Mouse Neuroblastoma (Neuro2A)
Treatment
Serum deprivation (0.1%) for 24 hrs
Blocking step
BSA as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 28°C

Abcam user community

Verified customer

Submitted Mar 25 2015

Abcam has not validated the combination of species/application used in this Abreview.
Application
Western blot
Sample
Apteronotus leptorhynchus Tissue lysate - whole (Brain)
Gel Running Conditions
Reduced Denaturing (4-15%)
Loading amount
50 µg
Specification
Brain
Blocking step
Milk as blocking agent for 14 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 4°C

Abcam user community

Verified customer

Submitted Nov 27 2012

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Rat Tissue sections (Brain)
Specification
Brain
Fixative
Formaldehyde
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: citrate buffer
Permeabilization
No
Blocking step
BSA as blocking agent for 10 minute(s) · Concentration: 1% · Temperature: 25°C

Dr. R Berahovich

Verified customer

Submitted Dec 01 2011

Abcam has not validated the combination of species/application used in this Abreview.
Application
Immunohistochemistry (Frozen sections)
Sample
Apteronotus leptorhynchus Tissue sections (Spinal cord, brain)
Specification
Spinal cord, brain
Fixative
Paraformaldehyde
Permeabilization
Yes - 0.3% Triton X-100
Blocking step
3% sheep serum, 1% BSA, 1% teleostean gelatine in PBS as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 3% · Temperature: 24°C

Dr. Ruxandra Sirbulescu

Verified customer

Submitted Nov 14 2011

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Flow Cytometry
Sample
Mouse Cell (Spleen)
Specification
Spleen
Preparation
Cell harvesting/tissue preparation method: 4% PFA was used to fixed the cells for 30 mins at room temperature.
Sample buffer: 1% BSA PBS was used as wash buffer
Fixation
Paraformaldehyde
Permeabilization
Yes - 0.5% Triton X-100

Abcam user community

Verified customer

Submitted Jul 20 2011

1-10 of 24 Abreviews

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