Anti-FOXC2 antibody - ChIP Grade (ab5060)

Tissues we tried (mouse tissues):
Venous valve sections
Lymphatic valve sections
Fixation:
Fresh frozen sections (unfixed) and then fixed in acetone for 10 min at -20C.
Also tried sections fixed in 1% PFA
Wash buffer 1xPBS plus 0.25% TX100
Block: 1% Normal Donkey serum in 1xPBS plus 0.25% TX100 for 30 min
Dilution used for FoxC2 was 1:150
Time: Tried 2 hrs with primary antibody as well as overnight at 4C
Secondary antibody: Donkey anti-goat conjugated with Cy3 for 30 minutes
The staining with fresh frozen sections that were acetone fixed used to work for us but no longer works with this new batch of antibody.
We are open to all suggestions

ANSWER:

Thank you for your reply.

We do not batch test ab5060 in IHC on frozen sections, we only guarantee it to work in IHC-P, ChIP and western blot. Therefore that would explain why you are not getting similar results, as we do not test for this application.

Were you seeing weaker signal than before? If so have you tried to increase the primary antibody concentration? Or were you seeing high background?

Have you tried the antibody in any of the test of the guaranteed applications? If so, does the antibody work in those applications?

We use the FoxC2 antibody from your company, Catology # ab5060. The first batch we received in 2008 worked fine for us. When we were low, we ordered it again. On , we received one vial (lot #GR29681) . We have tried to do immunohistochemistry with it several times since receiving it, but have not been able to get the results we are used to. We are using the same mouse tissues and parameters for staining, so should get similar results.
Have there been any issues with this lot #? Is there any way to get a fresh aliquot?

ANSWER:

Thank you for contacting Abcam.

The antibody is covered under our Abpromise for six months and is guaranteed to work in WB, ChIP and IHC-P on mouse and human samples. Since you ordered it in July last year, that is outside the six month guarantee but in this case I can make an exception and say that if we cannot resolve the issue you are having with the antibody then I send a replacement antibody.

To be able to better assist and see if there are any protocol suggestions I can make, can you please send me a copy of the protocol you are using, including species/cell types, primary antibody concentrations and incubations time, any antigen retrieval performed.

I look forward to your reply and helping you to resolve this issue.

The researcher is using ab5060 to stain human lymphedema sections (formalin fixed paraffin embedded) and has already contacted us for technical help. Dr Spotswood has suggested changing the pH of the antigen retrieval buffer to pH 9 but this has not changed the problem. The researcher has tried both IF and chromogenic detection with an HRP conjugated antibody. Incubation: overnight at 4C 1:200. no permeabilising agent.

ANSWER:

I have been able to open the reference Dagenais SL et al. Foxc2 is expressed in developing lymphatic vessels and other tissues associated with lymphedema-distichiasis syndrome. Gene Expr Patterns 4:611-9 (2004) which has used the antibody in immunohistochemistry and I hope the following detailed protocol will help you:

"Five micron paraffin sections were de-paraffinized with xylene and then re-hydrated in decreasing ethanol solutions according to standard procedures. Cryosections were incubated 10 min in acetone at ?20 °C and then air dried 15 min. Sections were washed three minutes in PBS before endogenous peroxidase activity was quenched by incubation for 20 min in 1.5% hydrogen peroxide in methanol. After one three-minute wash in PBS, antigen retrieval was performed on paraffin sections. ...For Foxc2... we used high temperature retrieval by incubating 10 min in boiling 0.01 M citrate buffer (pH 6.0), which was followed by cooling for 20 min at room temperature in the acid bath. Sections were washed for 3 min in PBS and then blocked for 10 min with TNB buffer (TSA" Fluorescence Systems) containing 0.1% Triton X-100 (t-Octylphenoxypolyethoxy-ethanol; Sigma, St Louis, MO, USA). Incubation with primary antibodies was 1 h at room temperature in a humidified chamber. Incubations with secondary and tertiary antibodies were 30 min in a humidified chamber. For protein signal amplification, sections were incubated 10 min with Fluorescein Tyramide (TSA" Fluorescence Systems). All incubations were followed by 3× 3-min washes in PBS containing 0.05% Tween 20 (Sigma). After a final series washes, sections were counterstained with DAPI (4?,6-Diamidino-2-phenylindole) in Vectashield mounting medium (Vector Labs, Burlingame, CA, USA). Signals were visualized with a Zeiss Axiophot Fluorescent microscope and imaging was performed using the Olympus DP70 Digital Camera System. Standard protocols were followed for Hematoxylin & Eosin staining."

Please do not hesitate to contact us if you still experience problems with this protocol,

Thank you for sending me a new vial of the FoxC2 antibody. I've tried it both with immunofluorescence and western blot and it works well. Thank you.

ANSWER:

I am delighted to hear you are satisfied with the replacement vial sent following your problem reported on the 3rd March; thank you for taking the time to let me know.

Please consider submitting a review and in return we will award you 50 points with the Abcam Loyalty Scheme which can be redeemed on Amazon gift vouchers.

Thank you again for your kind e-mail,

Customer is using this antibody in IHC-P with mouse gut and mesentary sections. Microwave antigen retrieval as recommended. She used DAB detection. However, saw staining in the cytoplasm as well as the nucleus. Has this been reported before?

ANSWER:

Thank you for your phone call. Originally, this product was released as a fast-track antibody and recently we have upgraded it. One of our customers has tested and used it successfully for application in IHC-formalin-fixed paraffin-embedded sections. I don't know if what you are seeing has been reported before. You may want to refer to the following publication, the authors used ab5060 in IHC:

Dagenais SL et al. Foxc2 is expressed in developing lymphatic vessels and other tissues associated with lymphedema-distichiasis syndrome. Gene Expr Patterns 4:611-9 (2004).

Please let me know if you require further assistance.