Anti-pan Keratin antibody [80] (ab8068)

I am looking to get some prepared control slides for the Anti-pan Keratin antibody [80] (ab8068) on your website. The overview of your antibody product suggests that the positive control is Human Callus or keratinocytes. I am looking to purchase prepared slides of the suggested control and was curious if you offered such a product or could point me in the right direction? Additionally, how much would these slides possibly cost?   Thank you for your time and assistance.    

ANSWER:

Thank you for contacting Abcam. I have looked through our catalogue to see if we have any prepared slides of human callus or keratinocytes. I have not been able to find anything that matches your requirements, but I have placed a link below that will show all of the prepared slides that we do have: www.abcam.com/index.html?pageconfig=catalog_byclass&intClassID=4186&strStartChar=A,B I also did a quick search of other companies to see if they had anything that matches your requirements but I could not find anything. Please let me know if there is anything else I can help with.  

I came across primary antibody ab51054. Has this antibody ever been tested for its cross-reactivity against pig??? Thank you so much for all your help.

Sincerely,

ANSWER:

Thank you for your question.

We do not have any information about cross reactivity with pig.

Please do not hesitate to contact us in case of any additional questions or remarks.

Thank you so much for the quick reply. I have another question.

For my particular experiment I am co-culturing porcine periodontal ligament epithelial cells with mouse fibroblast cells. I need a primary antibody to distinguish these two different cells from one another. (mouse fibroblast cells have green fluorescent protein so double immunostaining is not necessary).

Could you please recommend me with a primary antibody (preferably host animal to be a rabbit please) for this co-culture experiment. (my main goal is to distinguish two cells from each other).

Thank you so much for all your help.

Sincerely,

ANSWER:

Thank you for getting back to me and for explaining a little bit more about your experiments.

It may be worth applying antibodies which recognize pig albumin or serum (ab79960, ab50337, ab79957). Unfortunately, currently we do not have many antibodies specific for porcine proteins. The majority of our antibodies has been routinely tested in human, mouse and rat.

You could use antibodies against mouse MHC (class I) to detect and distinguish mouse cells from porcine cells. We have different clonality antibodies against mouse MHC class I, please have a look and use our new search engine. You can also filter through the search results by different criteria (such as application, reactivity, clonality):

http://www.abcam.com/

I hope this helps and if I can assist further, please do not hesitate to contact me.

To Whom it May Concern:

Hello,I purchased Mouse monoclonal [80] to pan Keratin (ab8068) few months ago and I've used it to immunostain my porcine periodontal ligament epithelial cells.

However, I was not able to stain my cells at all.

I've used

--4% Formaldehyde as fixitive

--10% goat serum in 3% BSA as a blocking solution

--Mouse monoclonal [80] to pan Keratin (ab8068) as a primary antibody

--Alexa fluor 546 goat-anti mouse as a secondary antibody

--0.05% Triton x-100 to permeabilize cells

Also, I was wondering if you can recommend another cytokeratin primary antibody for co-culture experiment consisting of porcine periodontal ligament epithelial cells and mouse fibroblast cells (I prefer host animal to be a rabbit please).

Thank you so much for all your help.

Sincerely,

ANSWER:

Thank you for contacting Abcam Technical Team and for taking the time to provide some useful details of the experiments. I am very sorry to hear that you are having problems with this product.

As our Abpromise indicates, in the event that a product is not functioning in the applications/species cited on the product data sheet (and the problem has been reported within 6 months of purchase) we will happily offer a credit note/refund to the value of the product purchased.

This antibody has been tested in human, mouse, rat as the datasheet indicates, however no data available yet regarding porcine tissue. It does not necessary mean that ab8068 is unsuitable in this species but it has not been characterized yet for cross-reaction.

Although, you have kindly provided some details, it would be much appreciated if you could give me more information about the staining (such as dilution/concentration with the primary antibody, blocking time and temperature, positive control etc) and complete the following form (attached as a word document). I could take a look at the protocol (if you wish) to see if any tips/recommendations can be made to improve the signal.

Thank you for your understanding and co-operation in this matter. I look forward to hearing from you soon and resolving this issue as soon as possible.

Do you have any information on using a mouse monoclonal on mouse tissue?

ANSWER:

Thank you for your enquiry.

When conducting IHC, it is not advisable to use antibodies which were raised in the same species as the target sample e.g. a mouse mAb against mouse sample. The secondary Ab may recognise endogenous IgG in your sample tissue as well as recognising the primary Ab, leading to non specific staining and a high background.

IHCWorld.com describes an additional blocking step using unconjugated Fab' fragment anti-mouse IgG to block endogenous IgG present in the tissue sample. Please see the following link for further information: http://ihcworld.com/_protocols/general_IHC/immuno_mom.htm

In addition, kits such as Dako’s "Animal Research kit" (ARK) allows the direct biotinylation of the primary Ab being used, negating the necessity for a secondary Ab and reducing the potential for non specific staining. The following paper (Van der Loos et al 2000) gives a summary of the use of these types of kit: http://www.jhc.org/cgi/content/full/48/10/1431

I hope this information helps, please do not hesitate to contact us if you need any more advice or information.