Anti-MHC Class II antibody [MRC OX-6] (ab23990)

Will this antibody recognize the D, B, and Q haplotypes?

ANSWER:

Thanks again for your call last week amd for your question about this antibody. I sincerely apologize but I just received further information from the lab, and the last email I sent you contained misinformation. This antibody has actually been tested in several strains of mice, and it does not appear to be reactive with the D,B, or Q haplotypes. I found an order assume is your order. If you have already opened this antibody, I can issue an immediate refund. If you have not opened the vial I can exchange it for a different MHC class II antibody or you can just return it for a refund. Again, I am very sorry for this error, and I look forward to hearing from you so we can resolve this promptly. Please let me know if you have any questions or if there is anything else that we can do for you.  

Will this antibody recognize the D, B, and Q haplotypes?

ANSWER:

Thank you for your call yesterday and for your patience while I have been in touch with the lab. I have attached a document that shows what strains have been tested with ab23990. According to the lab, we have not tested C57/Bl6, however every strain tested was positive. Since this antibody recognizes a monomorphic determinant, they are confident that it will work with the other strains as well. I hope this information will be useful, but if you need any further information please let me know and I'll be happy to help.    

Thanks for your reply, carefully read manuals of the two products ab8351, ab23990, I noticed about the following instructions: ‘ab23990--This antibody recognises a monomorphic determinant of the rat I-A antigen. ab8351--Reacts with monomorphic determinant on beta chain of RT-1B molecules’. Therefore, I have the following questions: 1, Whether the rat I -A antigen and RT-1B molecules are the same protein molecule or not?

2, As described in the literature (Immunogenetics. 2001 Sep;53(7):520-42), the rat MHC Class Ⅰ genes, which occur in the RT1-A and RT1-C/E/M region, are usually divided into the class Ia and class Ib subfamilies, and the rat MHC Class Ⅱ genes occur in the RT1-B/D region. So whether rat I-A antigen means that it is the Protein product of the rat MHC Class Ia gene? If the answer is yes, the ab23990 do not recognize MHC Class Ⅱ but recognize MHC Class Ⅰ.

So before I buy the product, I want to understand the two questions. I am waiting for your answers, and thank you very much. Hope for reply as quickly as possible. Thanks.

ANSWER:

Thank you for your patience.

I have received the following explanation from the lab that will hopefully help to answer your questions:

Please see below for a breakdown of rat and mouse MHC molecules.

Rat MHC Class II molecules are RT1.B, RT1.D and RT1.H. Class I molecules are simply RT1.

Mouse MHC class II molecules are I-A and I-E which happen to be very similar to the rat RT1.B and RT1.D but are named differently in each species.

ab23990 is described as mouse anti-rat MHC Class II RT1B, but also recognizes mouse MHC class II I-A in mice with haplotypes k and s.

And ab8351 is reacting with RT1B.

Regarding your question 1: The antigens being referred to are incorrect. Rat MHC Class II molecules are RT1.B, RT1.D and RT1.H. There is no rat I-A or I-B.

Regarding question 2: The antibody ab23990 is not targeted against MHC Class I molecules.

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

I am currently using MHC II (ab23990) on 10 um-thick frozen rat brain sections for immunofluorescence. Before adding this antibody I fix with 4% PFA (10min) and then permeabilize with 0.25% TTX-100 before blocking for 1 hr. I am not entirely convinced with the results, which may be related to the permeabilization step. I do understand that TTX-100 may be too harsh. I am somewhat confused as to whether I should permeabilize the section or not given that MHC II is located on endosomal/lysosomal membrane. Therefore, I would need to permeabilize to allow intracellular entry of antibody but at the same time wouldn't that also damage endosomal/lysosomal membrane where MHC II is located?

*My question: Is it best to permeabilize the cells for MHC-II for immunofluorescence?

ANSWER:

Thank you for your enquiry.

Permeabilisation is normally required for intracellular Flow Cytometry as this involves whole cells. However, for immunohistochemistry (whether using fluorescence or HRP), the cells have already been sliced, so further permeabilisation may not be required. We generally recommend acetone fixation as PFA can sometimes be problematic.

Would it be possible to try this antibody without permeabilisation as this is likely to be the cause of the issues that you are experiencing?

Please find below our recommended protocol for use with frozen sections: http://www.abdserotec.com/support/recommended-protocols-650.html