Anti-Melanoma antibody [HMB45 + M2-7C10 + M2-9E3] (ab732)

Thank you for contacting Abcam.
I have attached the Abcam, mouse on mouse staining protocol to see if that can give you any suggestions. If you can send me a copy of the protocol you are using, then I would be happy to see if there are any suggestions I can make.
I look forward to your reply.

I am sorry to confirm we do not currently have fluorochrome conjugated Anti-Melanoma antibody [HMB45 + DT101 + BC199] (ab732) on our catalog.


However, you may like to consider either using a secondary antibody (https://www.abcam.com/index.html?pageconfig=productmap&cl=918) or conjugating the antibody yourself using our EasyLink antibody coniugation kits suitable for fluorochrome detection methods.

EasyLink antibody conjugation kits provide a simple and quick linking process of your antibody with the advantage of one-step antibody labeling method with no separation steps. You can find more information about our EasyLink and procedures by clicking the following link:
https://www.abcam.com/index.html?pageconfig=resource&rid=13148#1

Although we do not have EasyLink antibody conjugation kit specifically for Alexa 647, we do have EasyLink kit for dyes with similar emission and excitation wavelengths as Alexa647: APC-Cy5.5, Cy5.5 and APC-Cy7. You can find them on the table at the bottom page of the link above, where you can also find the references of the different kits we dispose (you just have to click on them to have more information). The differences between those kits is the amount of reaction allowed.

Our Melanoma Cocktail is HMB45[HMB45] + two different clones of MART-1[M2-7C10] + [M2-9E3].
HMB34 isotype: IgG1/kappa and both clones of MART-1: IgG2b + IgG2b
Epitope/antigen: HMB45 and MART-1

Thank you very much for contacting us with your enquiry.
I am sorry to hear that there has been some variability with ab732. We don't have any other recent reports of the antibody not working, and there haven't been any changes to the antibody, so I'm not sure if this is a general problem or if it a faulty vial. The new vial that you have ordered is from a different production batch than the problematic one, so the results may improve using this vial.
Regarding the problematic vial, we do fully guarantee this antibody to work in the tested species and applications listed on the datasheet. Could you send me some details about the sample and protocol used with this antibody? Were the same samples and protocol used with the old and new vials? Do you have your order number for the vial that didn't work? I can arrange a credit or refund if you would like.
I look forward to hearing from you. Please let me know if you have further questions or if there is anything else that we can do for you.

Thank you for your enquiry. I am pleased to provide the following suggested positive controls for these antibodies. I have mainly chosen tissue that has been tested succesfully and shown in an image on the datasheets: 1.Anti-ErbB 2 antibody (ab2428) Human breast cancer 2.Anti-Progesterone Receptor antibody [SP2] (ab16661) Human breast carcinoma 3.Anti-Estrogen Receptor antibody [SP1] (ab16660) Human breast carcinoma 4.Anti-Myeloperoxidase antibody (ab45977) Human spleen tissue 5.Anti-TdT antibody (ab85148) Human testis 6.Anti-TMEM16A antibody (ab53212) Human liver 7.Anti-NSE antibody [EPR3377] (ab79757) Human brain tissue 8.Anti-CD23 antibody [EPR3617(IHC)] (ab92495) Human tonsil 9.Anti-CD10 antibody [56C6] (ab951) Lymph node tissue or human breast microepithelium 10.Anti-Melanoma antibody [HMB45 + DT101 + BC199] (ab732) Murine melanoma metastasis tissue 11.Anti-CD79a antibody [HM47/A9] (ab3121) Human Lymphoid tissue, tonsil 12.Anti-CD45RO antibody [UCHL-1] (ab86080) Human Tonsil, Thymus 13.Anti-CD45 antibody (ab10559) Human Tonsil tissue 14.Anti-CD30 antibody [CON6D/ C2] - BSA and Azide free (ab76518) Human Hodgekins lymphoma tissue 15.Anti-CD15 antibody [(Workshop IV) (BRA-4F1) our designation 238)] (ab115993) Human Hodgekins lymphoma tissue Human spleen I am providing a list ofslides that we sell which I hope will be helpful to you. I can recommend to review the online datasheet for more details. Human brain: ab4305Brain (human): hippocampus normal tissue slides https://www.abcam.com/Brain-human-hippocampus-normal-tissue-slides-ab4305.html (or use the following: https://www.abcam.com/Brain-human-hippocampus-normal-tissue-slides-ab4305.html). Human lymph node: ab4350Lymph node (human) normal tissue slides IHC-P, ISH https://www.abcam.com/Lymph-node-human-normal-tissue-slides-ab4350.html (or use the following: https://www.abcam.com/Lymph-node-human-normal-tissue-slides-ab4350.html). Human testis: ab4373Testis (human) normal tissue slides IHC-P, ISH https://www.abcam.com/Testis-human-normal-tissue-slides-ab4373.html (or use the following: https://www.abcam.com/Testis-human-normal-tissue-slides-ab4373.html). Human liver: ab4348Liver (human) normal tissue slides IHC-P, ISH https://www.abcam.com/Liver-human-normal-tissue-slides-ab4348.html (or use the following: https://www.abcam.com/Liver-human-normal-tissue-slides-ab4348.html). Human spleen: ab4372Spleen (human) normal tissue slides IHC-P, ISH https://www.abcam.com/Spleen-human-normal-tissue-slides-ab4372.html (or use the following: https://www.abcam.com/Spleen-human-normal-tissue-slides-ab4372.html). I am sorry we do not have slides forthe following: Human tonsil Hodgekins lymphoma tissue Mouse melenoma tissue I hope this will be helpful to you. If you have any further questions,please do not hesitate to contact us.

Thank you for your patience while I was looking into this. I have heard back from the lab and they have told me that ab732 was tested using the DIVA decloaking agent (http://biocare.net/wp-content/uploads/DV2004X.pdf) which is at pH.6.2. Also for the refrence of the antibody being tested in IHC paraffin embedded tissue, the only ones I can find of the antibody beind used on mouse tissue and/or on paraffin embedded sections are; Azarova AM et al. Roles of DNA topoisomerase II isozymes in chemotherapy and secondary malignancies. Proc Natl Acad Sci U S A 104:11014-9 (2007). Jungbluth AA et al. A103: An anti-melan-a monoclonal antibody for the detection of malignant melanoma in paraffin-embedded tissues. Am J Surg Pathol 22:595-602 (1998). If there is anything else I can help you with, please let me know.  

Thank you for contacting Abcam. The 3 clones in the ab732 cocktail do recognize mouse tissue. The IHCP protocol that was used to test the antibody was optimized to human tissue and the following protocol was used: Pretreatment Protocol Heat Retrieval Method: Retrieve sections under pressure using BIOCARE's Decloaking Chamber, followed by a wash in distilled water. Alternatively, steam tissue sections for 45-60 minutes. Allow solution to cool for 20 minutes then wash in distilled water. Protein Block: Incubate for 10-15 minutes at RT with BIOCARE's Background Sniper. Primary Antibody: Incubate for 30 minutes at RT. Probe: Incubate for 10 minutes at RT with a Probe. Chromogen: Incubate for 5 minutes at RT when using BIOCARE's DA Polymer : Incubate for 10 minutes at RT with a Polymer. If there is anything else I can help with, please let me know.

Thank you for your enquiry. You would not want to use the citrate buffer for antigen retrieval on your samples when using this antibody cocktail. You can use 1mM EDTA pH 8.0, and you should determine the optimal heating time for your sample. A good place to start would be 10-20 minutes. I hope this information helps, please do not hesitate to contact us if you need any more advice or information.