Anti-ICAM1 antibody (Biotin) (ab7815)

Dear Sir/Madam

I would like to ask if antibodies with cat no ab7815 and ab33451 are able to use for IHC staining in paraffin embedded tissue.

Thank you very much.

Regards,
Kay

ANSWER:

Thank you for contacting us.

ab7815 and ab33451 have been tested with Frozen sections only and we do not have any empirical data to support their performance with paraffin tissues.

I have found the following ICAM1 and Monocyte + macrophage antibodies which have been tested in IHC-P and might be of your interest.

Anti-ICAM1 antibody:

ab2213 ab109361 ab33894. They are not biotin labelled.

Anti-Monocyte + Macrophage antibody


ab51934. React with human.



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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I have a reply from the customer. Regarding the IHC testing, I was under the impression that they used paraffin sections, however, they used sections that were slightly fixed, impregnated with Sucrose and frozen to cut on a freezing microtome. Can you please take a look at the IHC form again with this information in mind? Regarding the suggestions, they do not have a skin biopsy available for a positive control. They did buy the antibody for IHC and only tried it in WB after it did not work in IHC, so they do not want to try the WB again if they do not have to.



Do you have suggestions for the antibody in IHC?

Thank you,

ANSWER:

Thank you for your email.

I have a couple recommendation for your customer when using IHC-Fr.

1.) I strongly suggest to run a positive control. ICAM1 belongs to the homing receptors and to my knowledge should not be expressed in brain. These homing receptor are expressed in site with inflammation to accumulate lymphocytes. If treated skin biopsies are not available, lung or bone marrow might be suitable.

2.) According to the protocol your customer send us, the mouse brain is fixed twice: perfusion fixed and fixed for 4days after. This strong (double-)fixation can lead to confirmation changes in the target protein which renders the antibody unable to recognize the target protein.
I recommend to try various antigen retrieval methods. This has to be tested experimentally.

3.) Your customer is correct, this antibody is labeled with biotin. Without using a secondary antibody there is less amplification of the signal and I therefore suggest to dilute the antibody less and to try 1:50.

I hope these tips can help improve the result. Please do not hesitate to contact me again with any further questions.

Good morning,

I had a customer contact me regarding some problems with AB7815 in WB and IHC (no signal). I realize that this antibody is not longer validated for paraffin sections, but the customer also tried it in WB without success. This item was ordered on PO xxxx. Can you please take a look at the customer's protocols attached?



Thank you



Kind regards

ANSWER:

Thank you and your customer for taking the time to complete our questionnaire and contact us. I am sorry to hear your customer has had difficulty obtaining satisfactory results from this antibody. I can confirm that ab7815 is covered by our Abpromise guarantee when used in WB.

The details your customer has kindly provided will enable us to investigate this case for your customer and this is also helpful in our records for monitoring of quality.

Reviewing this case, I would like to offer some suggestions to help optimise the results from ab7815:

1.) I suggest use a positive control: Fresh skin biopsy (small fresh skin biopsy incubated for 4 hours with TNF (1 ng/ml), IL-1 (100 u/ml) or LPS (1 ìg/ml) in tissue culture medium at 37oC).

2.) I recommend to use reducing and denaturing conditions: add SDS to the lysis puffer and add beta mercaptho-ethanol to the loading buffer.

3.) Blocking in BSA can significantly increase the signal of some antibodies. Therefore I suggest to use 5% BSA as blocking agent.

4.) Since there is no background I suggest to not use any blocking agent in the antibody solution and to dilute the antibody less. An other customer had good results with a dilution of 1:500.

We are happy to offer this technical support. In the event that a product is not functioning in the applications 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 hope this information is helpful, thank you and your customer for your cooperation. Should the suggestions not improve the results, please do not hesitate to contact me again with the further requested details.

Muchas gracias, cuando sepas algo nos comentas, y elegimos el que creamos pueda ser mejor.

ANSWER:

I can confirm that we didn't test the antibody (ab106777: Anti-VCAM1) in Rabbit tissue, as soon as we have further information we will update our on-line datasheet accordingly.

Muchas gracias, cuando sepas algo nos comentas, y elegimos el que creamos pueda ser mejor.

ANSWER:

Thank you for your interest.



The reactivity of this antibody (ab2213) with rabbit samples on Western blot application is probable but it has not been tested yet.



Hope this will be useful for you!