Anti-alpha smooth muscle Actin antibody [1A4] (ab7817)

Thank you for your help with this.



These are primary vascular smooth muscle cells brought in from a company. I'm using poly L lysine coated chamber slides with the cells at no more than passage number 5. When I put the cells on the slide I give them a day or so to settle down before starting the experiment. To harverst I simply tip the media off, wash and fix.



Since your e mail I have had success in staining for aSMA on a different batch of cells so I am now wondering about the validity of the original cells couldn't stain.



Any way, for now the issue seems to be fixed.



Whilst I am in contact with you however could I possibly ask you to look into something else for me?



I use a lot of abcam's P2X7 receptor antibody ab109246 but I cannot find anywhere the actual concentration of antibody so am unable currently to run a valid negative control of Rabbit IgG at the same concentration. Could you advise?



Many thanks

ANSWER:

Thank you for your email.

I am happy to hear that the problems are solved and the new experiment was successful.

The concentration of ab109246 is lot dependent.

I assume you are working with the lot number 1154802 (GR43315-5) or 1292653 (GR43315-6).

I can confirm they both have a concentration of 1.183 mg/mL.

I hope this information is helpful. Please do not hesitate to contact me again with any further questions. I wish you good luck with your experiments.

primary kidney cells in culture flask,chamber slides, than frozen, fixed Aceton
No results in ICC but works in tissue! Wrong sample? Is this prote. expressed in those cells?

ANSWER:

Thank you for your call.

In general, I would say that you should see a signal, however, I found that the developing state of the kidney and the localization of this protein is very important as well.

Please see the following publication and the attachment:

"in Rats, [...] During fetal life (17 days), alpha-SM actin also was expressed within juxtamedullary glomeruli. After 10 days of postnatal life, once glomerular development was completed, intraglomerular expression was no longer present. Peritubular capillaries expressed alpha-SM actin during early (fetal and neonatal) development, but not in the adult kidney. We conclude that 1) expression of alpha-SM actin in the developing kidney is delayed with respect to other vascular beds, 2) expression of alpha-SM actin follows the centrifugal pattern of nephrovascular development, and 3) glomerular and peritubular capillary expression of alpha-SM actin is a transient developmental phenomenon associated with active glomerulogenesis and capillary growth." Carey et al., "Expression of alpha-smooth muscle actin in the developing kidney vasculature", Hypertension. 1992 Feb;19(2 Suppl):II168-75..PMID: 1735573

For the localization, please see attachment.

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

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primary kidney cells in culture flask,chamber slides, than frozen, fixed Acetone
No results in ICC but works in tissue! Wrong sample? Is this prot. expressed in those cells?

ANSWER:

Thank you for contacting us.

I am sorry to hear you are experiencing difficulties with one of our products. We take product complaints very seriously, and investigate every product that we feel may not be performing correctly.

I am attaching our questionnaire so that we can gather further information regarding the samples tested and the protocol used. Once we receive the completed questionnaire, we will look at the protocol and see if there are any suggestions we can make that may improve the results. This information will also allow us to investigate this case internally and initiate additional testing where necessary. If the product was purchased in the last six months and is being used according to our Abpromise, we would be happy to replace or refund the antibody.


I look forward to receiving your reply.

Phone call requesting information on how to perform mouse on mouse staining potentially using anti-alpha smooth muscle Actin antibody (ab7817).

ANSWER:

Thank you fro contacting us.


I have found a couple of antibodies which may be more appropriate for the proceedureyouwishto perform. Ab5694 andab15734 are both rabbit polyclonals to alpha smooth muscle actin and has been shown to detect this in mouse samples and with paraffin embedded IHC tissue staining. These may be more appropriate as the background staining which can occur when using mouse-on-mouse staining can be avoided.


If you are interested in performing mouse-on-mouse staining, more information on how to achieve this can be found here:


http://www.abcam.com/index.html?pageconfig=resource&rid=11465


Additional information can also be found from IHC world here:


http://www.ihcworld.com/_protocols/general_IHC/immuno_mom.htm


I hope this information has been of help. If you have any further questions please do not hesitate to contact us again.

I am interested in purchasing a secondary antibody for use in immunohistochemistry that can work with the following primary antibodies from your company (ab24590, ab7817, ab955, and ab89064). I have searched the catalog and found ab6728 and ab6788 as possible candidates for this purpose. Could you kindly suggest on which one would work better with those primary antibodies for immunohistochemistry (IHC-P)?

Thank you,

ANSWER:

Thank you for contacting Abcam.

I feel that both products are suitable for IHC-P. Both have many specific references and in-depth scientific support data right on the datasheet. They differ mainly in the species in which they were raised and in the tags used for detection. Ab6728 is an HRP conjugate whereas Ab6788 is Biotin labeled. I believe that you should choose the secondary which has an associated detection system that you are most familiar with in case you should run into any staining issues.

I hope this information is helpful. Please let me know if you have any questions.