Unfortunately I must do double fluorescent immunostaining with two mouse monoclonal primary antibodies now. That is to say ,the primary antibodies are from the same animal origin as follows,

Mouse anti-A monoclonal antibody and Mouse anti-B monoclonal antibody.And the secondary antibodies are conjugated with FITC and TRITC respectively.As I know, these secondary antibodies could not differentiate IgG subclass.

My objective is to show the primary antibodies on one section simultaneously with secondary antibodies as above.

SO I meet with the most difficult type of double immunostaining. To avoid cross-reaction between the secondary antibodies and the primary antibodies, I should try my best and long for your help as well.

I've learned and tried the method of "post-fixation with paraformaldehyde steam". After incubation with the first kind of secondary antibody, incubate the section in PFA steam for several hours to inactivate the first kind of secondary antibody. Then add the second kind of primary antibody.

I succeeded once. But to be frankly, it is hard to control. I failed many times later. In my neighbor lab, a supervisor could manage this kind of double immunostaining well. Between incubation of the first kind of secondary antibody and the second kind of primary antibody, she use a kind of #161##176#Acidic Solution for antigen retrieval#161##177# to incubate section with application of microwave oven for 10 minutes.(But it is not citrate buffer as I know. And I#161#Ove tried citrate buffer and failed).However she would never tell me what components it has. (SAD.......it is one of her secrets).

I tried to find such a kind of acidic retrieval solution which could inactivate secondary antibody and retrieve antigen as well.Is there any kind of retrieval solution whose PH may be very low.And could be reused for many times as I know?

I wonder if you have such experience of double immunostaining? If so, could you kindly give me some advice?

Thank you very much!

 

Yichao WU, Ph.D Candidate

Email: yichaowu@hotmail.com

Research Institute of Nephrology

Jinling Hospital

Nanjing 210002 China