Question (15061) | Anti-MHC Class 1 H2 Db/H2-D1 antibody [27-11-13] (Phycoerythrin) (ab25547)

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Question

I want to use ab25547 and ab25588 which are primary antibodies to mouse MHCI proteins on fresh 10uM cryostat sections from mouse brain on slides that will be acetone fixed. Do you have a protocol for the acetone fixation step? I believe these antibodies are already conjucated to phycoerythrin. I have not used this before. Do I just do a protocol for primary antibody reaction, mount with gel mount (ab8214)then look under the scope with the FITC cube and look for yellow-orange cells? If so, do you have a protocol for this? Or should I use your ab8649 ABC kit? If so,then how? I guess I'm confused because the primary comes already conjugated to PE (probably for Facs).

Answer

Thank you for your enquiry. We do not have a protocol specifically for acetone fixation. However, may I recommend the excellent resource at IHC world. The following link details technical tips to consider when performing acetone fixation. http://www.ihcworld.com/_technical_tips/crystat_section_ihc.htm Both ab25547 and ab25588 are directly conjugated with phycoerythrin and therefore will not require a secondary antibody to detect them. You will not require ab8649. Yes, you are correct you will only need to perform the primary antibody incubation i.e. Omit steps 9-14 from the ABC Immunohistochemical Staining protocol located at the following link. https://www.abcam.com/index.html?pageconfig=view_protocol&pid=158 Phycoerythrin or PE is excited by a similar wavelength to FITC (488nm) but emits at 578nm as opposed to 519nm (FITC). Therefore Im not certain you would see much emission using a FITC filter. It may be necessary to modify the detection wavelength if possible to detect a wavelength of 578nm. I hope this information helps, please do not hesitate to contact us if you need any more advice or information.

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