Overview

  • Product name
    Anti-NCAM1 antibody [RNL-1]
    See all NCAM1 primary antibodies
  • Description
    Mouse monoclonal [RNL-1] to NCAM1
  • Host species
    Mouse
  • Specificity
    Neural cell adhesion molecule (NCAM). ab9018 reacts with three isoforms of NCAM: NCAM-120, NCAM-140, and NCAM-180.
  • Tested applications
    Suitable for: Flow Cyt, IHC-FoFr, ICC/IF, ICC, WB, IHC-Pmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Human
  • Immunogen

    This antibody was raised against the small lung cancer cell line NCI-H82.

  • Positive control
    • IHC-P: Mouse brain tissue. IHC-FoFr: Rat brain tissue. ICC/IF: Adult-derived human liver stem cells and hepatic stellate cells. Flow cytometry: HT1080 cells.
  • General notes
    Punctuate cytoplasmic staining is observed with ab9018. Some staining of red blood cells observed.

Properties

Applications

Our Abpromise guarantee covers the use of ab9018 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
Flow Cyt Use 2µg for 106 cells.

Paraformaldehyde or methanol fixed cells.

ab170190 - Mouse monoclonal IgG1, is suitable for use as an isotype control with this antibody.

IHC-FoFr 1/100 - 1/200. see detailed protocol
ICC/IF Use at an assay dependent concentration.
ICC Use at an assay dependent concentration.
WB 1/100 - 1/1000.
IHC-P Use at an assay dependent concentration.

Target

Images

  • ab9018 staining NCAM in Mouse brain tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with paraformaldehyde and blocked with 10% serum for 1 hour at 20°C; antigen retrieval was not performed. Samples were incubated with primary antibody (1/100) for 12 hours at 4°C. A Biotin-conjugated Rabbit anti-mouse polyclonal (1/200) was used as the secondary antibody.

    See Abreview

  • Immunocytochemical analysis of Adult-Derived Human Liver Stem Cells (ADHLSC) in the left image and Hepatic Stellate Cells (HSC) in the right image. ab9018 was used to label NCAM, which is expressed in the HSC. Cells were fixed in 3.5% paraformaldehyde for 15 minutes at room temperature. Blocking was with 3.3% hydrogen peroxide for 3 minutes. Permeabilization with D-PBS and 1% Triton X-100 for 10 minutes. Cells incubated with ab9018 at 1/100 for 1 hour. DAB staining. Counterstaining with Mayer's hematoxylin. 

  • Overlay histogram showing HT1080 cells stained with ab9018 (red line). The cells were fixed with 4% paraformaldehyde (10 min) and incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab9018, 2µg/1x106 cells) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 2µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in HT1080 cells fixed with methanol (5 min) used under the same conditions.

    Please note that Abcam does not have data for use of this antibody on non-fixed cells. We welcome any customer feedback.
  • ab9018 at a dilution of 1/500, staining NCAM (Alexa 488 secondary at 1/2000) on rat brain tissue (30µm thick coronal sections) in free floating IHC (see protocol link for detailed description). Images showing neuron body and processes: [A] 40x objective and [B] punctate cytoplasmic staining; 20x objective and [C] neuron + processes; x20 objective. No labeling observed following omission of primary antibody. Images coloured in Photoshop.

    NB: We recommend indirect amplification of immunofluorescence for ab9018

    Sections were viewed using an Axioplan 2 Imaging microscope (Imaging Associates) fitted with 10x, 20x and 40x Plan-Neofluorobjectives (Zeiss, Germany) and images were taken using a AxioCam Hrm digital camera (Zeiss, Germany) and AxioVision software (Imaging Associates).

References

This product has been referenced in:
  • Eguchi T  et al. Organoids with cancer stem cell-like properties secrete exosomes and HSP90 in a 3D nanoenvironment. PLoS One 13:e0191109 (2018). Read more (PubMed: 29415026) »
  • Wilson RJ  et al. Mitochondrial protein S-nitrosation protects against ischemia reperfusion-induced denervation at neuromuscular junction in skeletal muscle. Free Radic Biol Med 117:180-190 (2018). IHC-Fr ; Mouse . Read more (PubMed: 29432799) »
See all 16 Publications for this product

Customer reviews and Q&As

1-10 of 27 Abreviews or Q&A

Answer


The antibodies mentioned above are suitable for Rat as well as human samples. ab9018 is yet to be tested in IHC-Fr however as it is tested in IHC-FoFr so it is more likely to work.

The suitable secondary's are for ab4648, ab82259 and ab9018 are

https://www.abcam.com/rabbit-polyclonal-secondary-antibody-to-mouse-igg-hl-hrp-ab97046.html

https://www.abcam.com/goat-polyclonal-secondary-antibody-to-mouse-igg-hl-hrp-ab97023.html

for ab18258 it is

https://www.abcam.com/goat-polyclonal-secondary-antibody-to-rabbit-igg-hl-hrp-ab6721.html

for ab5392 it is;

https://www.abcam.com/goat-polyclonal-secondary-antibody-to-chicken-igy-h-l-hrp-ab97135.html

The protocols are:

https://www.abcam.com/index.html?pageconfig=resource&rid=11417

https://www.abcam.com/index.html?pageconfig=resource&rid=11383

https://www.abcam.com/index.html?pageconfig=resource&rid=13046

IHC-Fr is immunohistochemistry on frozen section

IHC-FoFr is PFA perfusion fixed frozen sections.

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Answer

Thank you for contacting us.

I am sorry that this antibody did not perform as stated on the datasheet. As requested, I have asked our Finance department to issue a credit note for a refund for you.

Credit note ID: #####

The credit note may be used in one of the following ways:

(1) Redeemed against the original invoice if this hasn't already been paid.
(2) Held on the account for use against a future order.
(3) A full refund can be offered where no other invoices are outstanding.

Please contact your Finance department to confirm how you would like the credit note to be used and ensure it is not redeemed without your knowledge.

To specifically receive a refund please ask your Finance department to contact our Finance department at creditcontrol@abcam.com or by telephone using the information at the “Contact Us” link in the top right corner of our website.

The credit note ID is for your reference only, please refer to the credit note ID in any correspondence with our accounting department. We will send you the completed credit note by email or postal mail with the actual credit note number which will start with the letters CGB.

I hope this experience will not prevent you from purchasing other products from us in the future. Our Scientific Support team is always at your service should you require further expert advice

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Answer

Thank you for taking the time to contact me again and provide an update. I am sincerely sorry to hear you have also had difficulty obtaining satisfactory results from the replacement vial of antibody.

I appreciate your concerns, this is disappointing. Reviewing the details, I am sorry there are no further tips to provide on this occasion to help improve the results, so I am not able to suggest anything further. I can reassure you that your enquiries will be kept on our quality monitoring records for our ongoing reference.

I apologize for the inconvenience. In this case, I suggest it would be appropriate to provide a refund or a credit note for purchase of another product.

Thank you for your cooperation. I look forward to hearing from you with details of how you would like to proceed this time.

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Answer

Thank you for your message.

I fully understand your concerns and would like to provide some information that I hope will give some reassurance. I can confirmthat we monitor feedback closely on a weekly basis and we are not currently concerned about the general quality of this antibody or this batch. Regrettably, I can suggest you have received a bad vial on this occasion.

Therefore, I hope the replacement vial should work well. However, please note that the replacement will be covered by the 6 month guarantee should you encounter any problems. I would encourage you to contact me as soon as possible in this case.

I hope this will be helpful. Please do not hesitate to contact me if you have any further questions.

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Answer

Thank you for confirming these details and for your cooperation. The details provided enable us to closely monitor the quality of our products.

I am sorry this product did not perform as stated on the datasheet and for the inconvenience this has caused. As requested, I have issued a free of charge replacement with the order number 1143353.

To check the status of the order please contact our Customer Service team and reference this number.

With regards to suggests positive controls, I can confirm that rat brain tissue should be a suitable positive control for this protein. There are some images from IHC staining of rat brain on the datasheet, also mouse brain. HT1080 cells woudl also be suitable.

According to the Unigene EST expression profile, NCAM is expressed in brain, adrenal gland, ear, larynx and lung, and to a lower extent in many other organs:


http://www.ncbi.nlm.nih.gov/UniGene/ESTProfileViewer.cgi?uglist=Hs.503878

Please note that the free of charge replacement vial is also covered by our Abpromise guarantee. Should you still be experiencing difficulties, or if you have any further questions, please do not hesitate to let us know.

I wish you the best of luck with your research.

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Question

Order Details
Antibody code: Anti-NCAM antibody [RNL-1] ab9018

Lot number: GR84221-1

General Information
Antibody storage conditions (temperature/reconstitution etc)
Short term 4C (1-2 weeks). Used most of the antibody during this period as I was trying various antibody dilutions and testing different tissue to see if I could get it to work.

Description of the problem (high background, low signal, non-specific satining etc.)

No staining was seen on any slides. I also tried a western blot using mouse muscle (NCAM a marker of muscle satellite cells), rat brain and human cell lines (see attached). No band was seen at the expected molecular weight of 140/180 kDa.

Sample (Species/Tissue/Cell Type/Cell Line etc.)

Mouse skeletal muscle tissue (paraffin embedded). Also rat brain as a control (frozen).

Fixation of sample (Ethanol/Methanol/Acetone/Paraformaldehyde/Other/Duration etc.)Paraffin embedded or frozen sections?
Paraffin embedded: Paraformaldehyde
Frozen: Ethanol

Antigen retrieval (Enzymatic method, Heat mediated technique etc.)

Tris-EDTA pH8 buffer using microwave technique.

Has citrate buffer retrieval been tried? What different time points were tried for optimization?
Citrate buffer hasn’t been tested, but I tried frozen rat brain tissue sections, and once again I could see no signal.

Permeabilization step


Blocking conditions (Buffer/time period, Blocking agent etc.)
Paraffin Embedded Mouse Tissue: Used mouse detection kit to eliminate background staining by the anti-mouse secondary antibody (MaxVision HM01-DS). Used blocking reagents, and 2 ry antibody supplied with kit and followed protocol.
Frozen Rat Brain Tissue: Used 1xCaesin (1 h 25C).
Western Blot: 5% Milk in TBS-Tw


blocking in 1xCaesin, same primary antibodies titrations and detection
I can recommend to try BSA or serum rather than Caesin to block. Changing blocking agent can sometimes help to improve the results.

Primary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step)

1:50, 1:100 and 1:200 in Dako Diluent (S0809) overnight at 4C.

Secondary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step)
Mouse Tissue: 2ry antibody supplied with mouse on mouse kit (manufacturer’s instructions).
Rat Tissue: 2ry Rabbit antimouse HRP (30 min, 25C). DAKO P0260
Western Blot: 2ry Goat antimouse HRP (1 h, 25C). Cell Signaling Tech. 7076

Detection method
IHC: DAB
Western Blot: ECL


Positive and negative controls used (please specify)

Rat brain tissue that was frozen. Once again, no signal was seen.
Negative Control: No primary antibody.

Optimization attempts (problem solving)
How many times have you tried the IHC?
5 Times with mouse muscle and rat brain

Have you run a No Primary control?
Yes

Is the current vial of secondary antibody working well with other primary antibodies?
Yes

Do you obtain the same results every time?
Yes


What steps have you altered?
Antibody concentration.
Frozen versus paraffin embedded tissue.
Rat Brain Control.
Western Blotting.
In all case no signal was seen.


Additional Notes
Although I wasn't restricted to satellite cells, I was expecting to see clear staining with the rat brain tissue. No signal at the correct molecular weight was seen on a Western Blot.

We would appreciate if you are also able to provide and image which woudl help us to assess the results
Image 1: IHC of paraffin embedded mouse skeletal muscle.
Imange 2: Western blot with NCAM1 (top half) and B-tubulin (bottom half).

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Answer

Thank you for taking the time to complete our questionnaire and contact us. I am sorry to hear you have had difficulty obtaining satisfactory results from this antibody.

The details you have kindly provided will provide us with vital information for our monitoring of product quality

I appreciate the time you have spent in the laboratory and understand your concerns. It is regrettable the results have not been successful. Reviewing the details, I am sorry there are no further tips to provide on this occasion to help improve the results. I can suggest you have regrettably received a bad vial.

I apologize for the inconvenience and am pleased to offer you a free of charge replacement or credit note in compensation.

In addition, I can suggest you may like to consider the following suggestions as a check for future experiments:

1. Antigen retrieval can sometimes require some optimization. I can recommend to try citrate buffer for 2, 5, 10 and 20 minutes, or enzymatic retrieval.

2. I can recommend to try BSA or serum rather than Caesin to block. Changing blocking agent can sometimes help to improve the results.

3. Are the current vials of secondary antibody working well with other primary antibodies?

4. I can suggest to consider Including a permeabilization step which would help to improve the results.

Thank you for your cooperation. I look forward to hearing from you with details of how you would like to proceed.

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Answer

Thank you for taking the time to contact us. I am sorry to hear you have had difficulty obtaining satisfactory results from this antibody.

I would like to reassure you that this antibody is tested and covered by our 6 month guarantee forIHC-P andIHC-FoFr and in mouse and rat samples. In the event that a product is not functioning in the tested applications and species cited on the product data sheet (and the problem has been reported within 6 months of purchase), we will be pleased to provide a credit note, free of charge replacement or refund.

I would like to investigate this particular case further for you, and also obtain more information for our quality monitoring records. In order to proceed with this, I have enclosed a technical questionnaire below. I would appreciate if you could complete this. It will help you put the information we require together very easily.

I would appreciate if youare also able to provide an image which would help us to assess the results.

Thank you for your time and cooperation. We look forward to receiving the completed questionaire.



Order Details
Antibody code:

Lot number:

Purchase order number
or preferably Abcam order number:


General Information
Antibody storage conditions (temperature/reconstitution etc)


Description of the problem (high background, low signal, non-specific satining etc.)

No staining was seen on any slides

Sample (Species/Tissue/Cell Type/Cell Line etc.)

mouse skeletal muscle tissue

Fixation of sample (Ethanol/Methanol/Acetone/Paraformaldehyde/Other/Duration etc.)Paraffin embedded or frozen sections?

Antigen retrieval (Enzymatic method, Heat mediated technique etc.)

Tris-EDTA pH8 buffer


Has citrate buffer retrieval been tried? What different time points were tried for optimization?

Permeabilization step


Blocking conditions (Buffer/time period, Blocking agent etc.)

blocking in 1xCaesin, same primary antibodies titrations and detection

I can recommend to try BSA or serum rather than Caesin to block. Changing blocking agent can sometimes help to improve the results.

Primary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step)

1:50, 1:100 and 1:200 in Dako Diluent (S0809) overnight at 4C

Secondary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step)


Detection method


Positive and negative controls used (please specify)

Rat brain tissue that was frozen and once again, no signal was seen

Optimization attempts (problem solving)
How many times have you tried the IHC?



Have you run a "No Primary" control?
Yes No


Is the current vial of secondary antibody working well with other primary antibodies?


Do you obtain the same results every time?
Yes No


What steps have you altered?


Additional Notes


We would appreciate if you are also able to provide an image which would help us to assess the results

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Answer

Thank you for contacting us.

I suggest having a look at some of the references for our NCAM antibodies such as ab9018, to see if this marker will be appropriate for what you are trying to do. It is not clear to me if NCAM is on all neurons or just a subset, though.

Click here (or use the following: https://www.abcam.com/index.html?datasheet=9018).

All other markers that I know of are intracellular.

While searching for marker candidates, I came across a paper which appears to have a good discussion of tissue dissociation techniques,

Stem Cells. 2007 Jun;25(6):1560-70.

Please do not hesitate to contact us if you need any more advice or information.

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Question
Answer

Thank you for contacting us. I am sorry that this antibody did not perform as stated on the datasheet. The above amount will be credited back to your credit card. To avoid confusion, please ensure your accounts department is aware of how the credit note is being used. Our accounting department can be contacted by email at us.credits@abcam.com or by telephone using the information at the Contact Us link in the top right corner of our website. Please refer to the credit note ID in any correspondence with our accounting department. The credit note ID is for your reference only and does not automatically guarantee the credit. I hope this experience will not prevent you from purchasing other products from us in the future. Our Scientific Support team is always at your service, should you require further expert advice.

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Answer

Thank you for sending those images. Comparing the images you sent and the images from the Allen Institute for Brain Science, see link below: http://mouse.brain-map.org/gene/show/17734 It would appear that your staining is similar to that theirs, maybe a little stronger but it all seems to be good (as long as I am reading you slides correctly and you were looking at the Cerebellum). To try and reduce the intensity of the staining, you could lower the primary antibody concentration to 1/150 and also block for longer. Please let me know if there is anything else I can help you with.    

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1-10 of 27 Abreviews or Q&A

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