Hi Funda, 

What an interesting question! Although for you it is probably more frustrating than scientifically inspiring! ;)

I have some experience with staining mouse tissue with mouse primary antibody, but those samples came from immun-defficient mice, so we had practically no problem with endogenous IgG. In the literature everyone recommends adding an extra incubation step with an unconjugated affinity purified F(ab) fragment anti-rabbit IgG (H+L) for 1 hr at room temperature, or overnight at 4°C. Abcam says to try this blocking antibody at 0.1 mg/ml first, then try to optimize its concentration. 

How does your no-primary control look like? How long is your blocking? This might sound radical but for us completely eliminating blocking resulted in a reduced background more than once, although not with identical sample-Ab species.

Best of luck, and please keep us updated on what works at the end!

Hi Funda, 

What an interesting question! Although for you it is probably more frustrating than scientifically inspiring! ;)

I have some experience with staining mouse tissue with mouse primary antibody, but those samples came from immun-defficient mice, so we had practically no problem with endogenous IgG. In the literature everyone recommends adding an extra incubation step with an unconjugated affinity purified F(ab) fragment anti-rabbit IgG (H+L) for 1 hr at room temperature, or overnight at 4°C. Abcam says to try this blocking antibody at 0.1 mg/ml first, then try to optimize its concentration. 

How does your no-primary control look like? How long is your blocking? This might sound radical but for us completely eliminating blocking resulted in a reduced background more than once, although not with identical sample-Ab species.

Best of luck, and please keep us updated on what works at the end!

Hello Funda,

I am doing IHC on mouse tissue as well. I am using a combination of 3% BSA in PBS for my blocking solution (1 hour incubation) and the primary antibody is also murine.

Incubation of the primary antibody is done overnight and then we wash several times with PBS before the secondary antibody is applied for 2 hours at RT, then again washed 3 times in PBS.

With this protocol we obtain very good results. But I am working with slices, maybe this is different to whole tissue stainings?

Good luck instead!

Dear Nora and Natascha

Thanks for your quick replies. I'll consider your advices absolutely.

Background staining will not make me discouragement :D 

Kind regards.

Hi Funda, you seem to have tried all the right things - sometimes there is no perfect solution. One possible solution, depending on how much antibody you have and what tissues you are staining, is to conjugate the primary to biotin. Then just use the avidin component of the ABC kit. This should eliminate background due to the secondary antibody. It isn't difficult but is time consuming and you'll have to test the antibody afterwards to ensure it still binds and to find the best concentration. 

Hi. Funda the way the tissue is prepared and processed may influence the background. Which tissues are you using?, What processing techniques are you using?, frozen or fixed tissues, paraffin embedded, etc. Are you preparation mounted on glass slides?, which type of slides sides??., negative charged?, are your tissues well washed when removed to remove left over blood which gives a lot of background?, which controls are you running?, do you get the same background on the controls??. Answering these questions may help you to find the solutions. Have you done tritiation and dilution curve of the antibody?.