SOX2 antibody - Embryonic Stem Cell Marker (ab15830)

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Anonymous Abreview submitted on 31 July 2009
Application:	Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
SOX2 antibody - Embryonic Stem Cell Marker (ab15830)
Sample	Mouse Tissue sections (embryonic stomach)
Specification	embryonic stomach
Fixative:	methacarn
Antigen retrieval step:	None
Permeabilization:	Yes - 0.3% tritonX-100
Blocking step:	Serum as blocking agent for  1 hour  · Concentration: 10 % · Temperature: 25°C
Primary Antibody
Dilution	1/50
Incubation time:	1 hour  Temperature: 25°C Diluent: 2% serum
Secondary Antibody
Name:	Non-Abcam Antibody was used: Pig anti Rabbit swine biotinylated Conjugation: Biotin
Dilution:	1/500
Additional Data
Rating	Good
Image	SOX2 antibody - Embryonic Stem Cell Marker for IHC-P (Mouse) Copyright image used under license

review by Carl Hobbs submitted on 2 September 2008
Application:	Immunocytochemistry/ Immunofluorescence
SOX2 antibody - Embryonic Stem Cell Marker (ab15830)
Sample	Mouse Cell (Cor1 Neural stem cells)
Specification	Cor1 Neural stem cells
Fixative:	Formaldehyde
Permeabilization:	Yes - 0.1% Tween
Blocking step:	BSA as blocking agent for  10 mins  · Concentration: 1 % · Temperature: rt°C
Primary Antibody
Dilution	1/500
Incubation time:	1 hour  Diluent: TBS/BSA/Tween/azide
Secondary Antibody
Name:	Non-Abcam Antibody was used: anti rabbit IgG Conjugation: Alexa Fluor® 594
Dilution:	1/1000
Additional Data
Rating	Excellent
Additional notes	Cells ( grown on coverslips in 4-well dishes) were fixed by adding an equal volume of 4%PFA to the culture medium for 10mins. Fluid was then decanted and replaced with 4%PFA for 10mins. Coverslips were then washed x3 in PBS and stored at 4C in PBS/azide for 3 days before immunostaining. I do not recommend this as a Standard Procedure as some Antigens become attenuated upon storage. Images captured using a manual Axioskop microscope, illuminated using an Xcite 120W light source. Colour balance was manually adjusted, using Photoshop "levels".
Image	SOX2 antibody - Embryonic Stem Cell Marker for ICC/IF (Mouse) Copyright image used under license

review by Sandra Pinho submitted on 6 March 2008
Application:	Immunocytochemistry/ Immunofluorescence
SOX2 antibody - Embryonic Stem Cell Marker (ab15830)
Sample	Human Cell (Neural Progenitors derived from H7 embryonic stem)
Specification	Neural Progenitors derived from H7 embryonic stem
Fixative:	Paraformaldehyde
Permeabilization:	Yes - 0.4% Triton X100 in PBS
Blocking step:	Serum as blocking agent for  30 mins  · Concentration: 10 % · Temperature: 25°C
Primary Antibody
Dilution	1/300
Incubation time:	16 hours  Temperature: 4°C Diluent: 2.5 % BSA + 0.05% Tween20 + 10% NGS /PBS
Secondary Antibody
Name:	Non-Abcam Antibody was used: Invitrogen Alexa Fluor 488 goat anti-rabbit IgG (H Conjugation: Alexa Fluor® 488
Dilution:	1/500
Additional Data
Rating	Excellent
Additional notes	Human embryonic stem cells (H7 cell line) upon differentiation towards the neural lineage by blocking the BMP signalling with noggin retain the expression of Sox2 like neural stem cells.
Image	SOX2 antibody - Embryonic Stem Cell Marker for ICC/IF (Human) Copyright image used under license

review by Carl Hobbs submitted on 3 December 2007
Application:	Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
SOX2 antibody - Embryonic Stem Cell Marker (ab15830)
Sample	Quail Tissue sections (Quail brain stem T/S)
Specification	Quail brain stem T/S
Fixative:	Formaldehyde
Antigen retrieval step:	Heat mediated - Buffer/Enzyme Used: Citric acid pH6 10mM
Permeabilization:	No
Blocking step:	BSA as blocking agent for  10 mins  · Concentration: 1 % · Temperature: RT°C
Primary Antibody
Dilution	1/600
Incubation time:	2 hours  Diluent: TBS/BSA/Azide
Secondary Antibody
Name:	Non-Abcam Antibody was used: Goat anti rabbit IgG Conjugation: Biotin
Additional Data
Rating	Excellent
Additional notes	An interesting protein: seems to be expessed in many developing/mature cells; neuronal and non-neuronal! Picture shows nuclear expression in embronic quail brain stem, ventricular region
Image	SOX2 antibody - Embryonic Stem Cell Marker for IHC-P (Quail) Copyright image used under license

review by Carl Hobbs submitted on 27 November 2007
Application:	Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
SOX2 antibody - Embryonic Stem Cell Marker (ab15830)
Sample	Dogfish/Catshark Tissue sections (Cochlear organ, I think)
Specification	Cochlear organ, I think
Fixative:	Formaldehyde
Antigen retrieval step:	Heat mediated - Buffer/Enzyme Used: Citric acid
Permeabilization:	No
Blocking step:	BSA as blocking agent for  10 mins  · Concentration: 1 % · Temperature: RT°C
Primary Antibody
Dilution	1/600
Incubation time:	2 hours  Diluent: TBS/BSA/Azide pH7.5
Secondary Antibody
Name:	Non-Abcam Antibody was used: Goat anti IgG Conjugation: Biotin
Additional Data
Rating	Excellent
Additional notes	I am not familiar with catshark/dogfish histology but this looks like part of a sound sensing organ. Please enlighten me! Abcam's TUJ1 ab18207 reagent beautifully identifies invaginating neuronal fibres of this organ (see my catshark TUJ1 review)
Image	SOX2 antibody - Embryonic Stem Cell Marker for IHC-P (Dogfish/Catshark) Copyright image used under license

review by Carl Hobbs submitted on 12 November 2007
Application:	Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
SOX2 antibody - Embryonic Stem Cell Marker (ab15830)
Sample	Zebrafish Tissue sections (adult zebra fish spinal cord T/S)
Specification	adult zebra fish spinal cord T/S
Fixative:	Formaldehyde
Antigen retrieval step:	Heat mediated - Buffer/Enzyme Used: citric acid pH6
Permeabilization:	No
Blocking step:	BSA as blocking agent for  10 mins  · Concentration: 1 % · Temperature: RT°C
Primary Antibody
Dilution	1/600
Incubation time:	2 hours  Diluent: TBS/BSA/Azide
Secondary Antibody
Name:	Non-Abcam Antibody was used: Goat anti rabbir IgG Conjugation: Biotin
Dilution:	1/600
Additional Data
Rating	Good
Additional notes	I am very surprised that this appears to be a "good" marker, for Zebrafish. I am not very familiar with the model, although I use it occassionally. The fact that this section is of adult spinal cord worries me....Why is the adult spinal cord positive for SOX2? I also have a picture of adult zebra fish skin, from the same test: this shows islands of cells that are positive for SOX2. If anyone has a knowledge of this in zebrafish, I would be grateful for advice. Please look in the Immuno pic gallery here for the picture: http://www.immunoportal.com/
Image	SOX2 antibody - Embryonic Stem Cell Marker for IHC-P (Zebrafish) Copyright image used under license
Abcam response	Many thanks for your comprehensive Abreview for ab15830 (SOX2 antibody - Embryonic Stem Cell Marker) in Zebrafish spinal cord. We would perhaps expect this antibody to detect Zebrafish SOX2 protein as the immunising peptide used to generate ab15830 has 90% identity and positivity with SOX2 Zebrafish protein (as stated on the datasheet). We welcome any feedback as to why this staining is observed in the adult Zebrafish spinal cord.

review by Carl Hobbs submitted on 5 November 2007
Application:	Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
SOX2 antibody - Embryonic Stem Cell Marker (ab15830)
Sample	Chicken Tissue sections (Brain embryonic day 7)
Specification	Brain embryonic day 7
Fixative:	Formaldehyde
Antigen retrieval step:	Heat mediated - Buffer/Enzyme Used: Citric acid pH 6 HIER
Permeabilization:	No
Blocking step:	BSA as blocking agent for  10 mins  · Concentration: 1 % · Temperature: RT°C
Primary Antibody
Dilution	1/600
Incubation time:	2 hours  Diluent: TBS/BSA/Azide
Secondary Antibody
Name:	Non-Abcam Antibody was used: Goat anti rabbit IgG Conjugation: Biotin
Dilution:	1/300
Additional Data
Rating	Excellent
Additional notes	This Ab reagent works very well, in chick, to my surprise. The ventricular region that is positive for Sox-2 ids sharply defined, in my picture.
Image	SOX2 antibody - Embryonic Stem Cell Marker for IHC-P (Chicken) Copyright image used under license

review by Carl Hobbs submitted on 5 November 2007
Application:	Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
SOX2 antibody - Embryonic Stem Cell Marker (ab15830)
Sample	Mouse Tissue sections (Embryonic day18 spinal cord, T/S)
Specification	Embryonic day18 spinal cord, T/S
Fixative:	Formaldehyde
Antigen retrieval step:	Heat mediated - Buffer/Enzyme Used: Citric acid pH6
Permeabilization:	No
Blocking step:	BSA as blocking agent for  10 mins  · Concentration: 1 % · Temperature: RT°C
Primary Antibody
Dilution	1/600
Incubation time:	16 hours  Diluent: TBS/BSA/Azide
Secondary Antibody
Name:	Non-Abcam Antibody was used: anti rabbit IgG Conjugation: Biotin
Dilution:	1/300
Additional Data
Rating	Good
Additional notes	A reagent that requires, in my hands, more fine-tuning to achieve an optimal signal-noise ratio, than many other antibody reagents. However, the nuclear positivity appears to conform with published data. Note that there is far less Sox2 positivity in the ventricular region of this mouse spinal cord: I assume that this is due to maturation of neurones/greatly decreased numbers of stem cells, at this late-developmental stage. I note a more dispersed positivity, outside of the ventricular region, than I noted in a rat embryonic day15 abreview.
Image	SOX2 antibody - Embryonic Stem Cell Marker for IHC-P (Mouse) Copyright image used under license

review by Carl Hobbs submitted on 23 October 2007
Application:	Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
SOX2 antibody - Embryonic Stem Cell Marker (ab15830)
Sample	Rat Tissue sections (T/S in situ E15 spinal cord)
Specification	T/S in situ E15 spinal cord
Fixative:	Formaldehyde
Antigen retrieval step:	Heat mediated - Buffer/Enzyme Used: citric acid pH6
Permeabilization:	No
Blocking step:	BSA as blocking agent for  10 mins  · Concentration: 1 % · Temperature: RT°C
Primary Antibody
Dilution	1/600
Incubation time:	1 hour  Diluent: TBS/BSA/Azide
Secondary Antibody
Name:	Non-Abcam Antibody was used: anti rabbit IgG Conjugation: Biotin
Dilution:	1/300
Additional Data
Rating	Good
Additional notes	I have tested only two anti-SOX2 Abs: this one is far superior/more reliable than the other. I see cytoplasmic positivity in some DRG cells (is this just because of inadequate dilution factor, or real positivity?). I also get nuclear positivity in rat embryo oesophagus/trachea epithelium: more pics posted here - http://www.immunoportal.com/index.php in the ImmunoHisto pic gallery. NB1: it is not a "fantastic" Ab reagent but it deserves to be a "very good" reagent, rather than just a "good" reagent. NB2: Nuclei in the ventricular region of the rat developing spinal cord (neural tube) are seen to be positive, in this picture. NB3: Some nuclei of cells in the rat svz of the lateral ventricle are also positive. This conforms to accepted knowledge
Image	SOX2 antibody - Embryonic Stem Cell Marker for IHC-P (Rat) Copyright image used under license
Abcam response	Many thanks for the detailed feedback you have sent for ab15830 (SOX2 antibody -Embryonic Stem Cell Marker) in IHC-P on rat spinal tissue.

Anonymous (click to contact this user) Abreview submitted on 2 May 2007
Application:	Western blot
SOX2 antibody - Embryonic Stem Cell Marker (ab15830)
Sample	Rat Cell lysate - whole cell (Brain, Liver)
Loading amount:	50 µg
Specification	Brain, Liver
Blocking step:	BSA as blocking agent for  1 hour  · Concentration: 5%
Primary Antibody
Concentration	0.3 µg/ml
Incubation time:	12 hours
Secondary Antibody
Name:	Abcam antibody: Rabbit IgG secondary antibody - H&L
Dilution:	1/2000
Detection
Detection method:	ECL+
Exposure:	4 minutes and 0 seconds
Bands:	Specific: 39 kDa
Positive Control:	NOne
Negative Control:	None
Additional Data
Rating	Excellent
Image	SOX2 antibody - Embryonic Stem Cell Marker for WB (Rat) Copyright image used under license

review by Heather Etchevers submitted on 22 March 2007
Application:	ChIP
SOX2 antibody - Embryonic Stem Cell Marker (ab15830)
Sample	Mouse Tissue lysate - whole (Embryonic day 13 whole head)
Specification	Embryonic day 13 whole head
Type:	Cross-linking (X-ChIP) - 0 Mins 0 Secs
Detection step:	Semiquantitative PCR
Positive Control:	ab8580 anti-Histone H3 (tri methyl K4) Tissue strongly expresses Sox2 and Pax6 proteins as well as target factor.
Negative Control:	ab5790 anti-Pax6 Rabbit anti-mouse IgG
Primary Antibody
Concentration	4 µg/ml
Incubation time:
Additional Data
Rating	Good
Additional notes	Classic X-ChIP protocol based on Abcam protocol; lengthening bead rinse steps to 10 minutes. We pre-absorbed antibodies for 2h at 4C onto 60 ul resuspended Protein A-agarose beads (Abcam ref 16-157) in 1 ml ChIP dilution buffer with 2% BSA, rinsing 1x 30 and 1x 5 minutes and resuspending after in 60 ul ChIP dilution buffer. In this image, the PCR amplicon concerns a predicted Sox2-binding site in the promoter of a putative transcriptional target, that was not predicted to also bind Pax6. The anti-Sox2 lane shows IP of this chromatin target, but the anti-Pax6 lane does not. It is also not IP'd by a non-specific rabbit polyclonal anti-mouse antibody ("IgG"). Anti-histone H3 ("anti-H3") confirms that the target is indeed transcribed in the embryonic mouse head at the time of sampling. Total input DNA also is amplifiable, as expected. However, the beads that were not pre-absorbed with an antibody ("no Ab"), also non-specifically precipitate amplifiable chromatin. This does not impinge on the efficacy of the anti-Sox2 antibody.
Image	SOX2 antibody - Embryonic Stem Cell Marker for ChIP (Mouse) Copyright image used under license
Abcam response	Thank you for a very thorough explanation of this well-controlled experiment.

review by Joana Neves submitted on 21 February 2007
Application:	Immunocytochemistry/ Immunofluorescence
SOX2 antibody - Embryonic Stem Cell Marker (ab15830)
Sample	Human Cell (HeLa cells CCL-2 transfected with chicken SOX3)
Specification	HeLa cells CCL-2 transfected with chicken SOX3
Fixative:	Paraformaldehyde
Blocking step:	Serum as blocking agent for  30 mins  · Concentration: 10%
Primary Antibody
Dilution	1/400
Incubation time:	1 hour
Secondary Antibody
Name:	Non-Abcam Antibody was used: Alexa-594 conjugated goat anti-rabbit Conjugation: Alexa Fluor® 594
Dilution:	1/400
Additional Data
Rating	Excellent
Additional notes	The chicken Sox3-GFP fusion gene was transfected in HeLa CCL-2 cells that were then probed by immunofluorescence with SOX2 or SOX3 antibodies.These experiments showed that anti-SOX2 antibody was unable to recognise the chicken SOX3 protein when expressed in HeLa cells. The expression of SOX3 was assessed by the concomitant detection of GFP in the same cells and also by immunofluorescence with the SOX3 antibody in parallel plates.
Image	SOX2 antibody - Embryonic Stem Cell Marker for ICC/IF (Human) Copyright image used under license

review by Joana Neves submitted on 20 March 2008
Application:	Immunocytochemistry/ Immunofluorescence
SOX2 antibody - Embryonic Stem Cell Marker (ab15830)
Sample	Chicken Cell (c-SOX3 trasfected HeLA cells CCL-2)
Specification	c-SOX3 trasfected HeLA cells CCL-2
Fixative:	Paraformaldehyde
Blocking step:	Serum as blocking agent for  30 mins  · Concentration: 10%
Primary Antibody
Dilution	1/400
Incubation time:	1 hour
Secondary Antibody
Name:	Non-Abcam Antibody was used: Alexa-594 Goat Conjugated anti-rabbit Conjugation: Alexa Fluor® 594
Dilution:	1/400
Additional Data
Rating	Excellent
Additional notes	This experiment was performed to show that SOX2 antibody did not recognize the SOX3 protein overexpressed in HeLa cells. Fixation was in PFA 4% in PBS for 15 min. Cells were washed in PBS 0.1% Tween for 10 min for permeabilization.
Image	SOX2 antibody - Embryonic Stem Cell Marker for ICC/IF (Chicken) Copyright image used under license

review by Joana Neves submitted on 19 January 2007
Application:	Immunohistochemistry (Frozen sections)
SOX2 antibody - Embryonic Stem Cell Marker (ab15830)
Sample	Chicken Tissue sections (Embryonic optic epithelium [A] and neural tube[B])
Specification	Embryonic optic epithelium [A] and neural tube[B]
Fixative:	Paraformaldehyde
Blocking step:	Serum as blocking agent for  1 hour  · Concentration: 10%
Primary Antibody
Dilution	1/400
Incubation time:	18 hours
Secondary Antibody
Name:	Non-Abcam Antibody was used: Alexa-594 conjugated goat anti-rabbit Conjugation: Alexa Fluor® 594
Dilution:	1/400
Additional Data
Rating	Excellent
Additional notes	SOX2 antibody stained the nuclei of the supporting cells in the basal layer of E17 cochlea and the ventricular zone of the neural tube on and E5 embryo. A, countersatined with DAPI and B, with Tuj1.
Image	SOX2 antibody - Embryonic Stem Cell Marker for IHC-Fr (Chicken) Copyright image used under license

review by Eumorphia Remboutsika submitted on 23 October 2006
Application:	Western blot
SOX2 antibody - Embryonic Stem Cell Marker (ab15830)
Sample	Mouse Cell lysate - whole cell (Cos7 transfected cells)
Loading amount:	50 µg
Specification	Cos7 transfected cells
Blocking step:	Milk as blocking agent for  12 hours  · Concentration: 10%
Primary Antibody
Dilution	1/750
Incubation time:	2 hours
Secondary Antibody
Name:	Non-Abcam Antibody was used: goat anti-rabbit IgG Conjugation: HRP
Dilution:	1/10000
Detection
Detection method:	ECL+
Exposure:	5 minutes and 0 seconds
Bands:	Specific: 35 kDa
Positive Control:	Observed band at around 35kDa in Sox2 transfected Cos-7 cells
Negative Control:	no observed bands in : untransfected Cos7 cells Sox1-transfected Cos7 cells Sox3-transfected Cos7 cells Sox9-transfected Cos7 cells
Additional Data
Rating	Excellent
Image	SOX2 antibody - Embryonic Stem Cell Marker for WB (Mouse) Copyright image used under license