I guess the cleanest way would be to directly conjugate your detection moiety (e.g. HRP) to your primary antibody. Any detection method that relies on detecting your rabbit antibodies by a secondary antibody recognizing rabbit immunoglobulins will also detect endogenous antibodies in rabbit spleen.

I guess the cleanest way would be to directly conjugate your detection moiety (e.g. HRP) to your primary antibody. Any detection method that relies on detecting your rabbit antibodies by a secondary antibody recognizing rabbit immunoglobulins will also detect endogenous antibodies in rabbit spleen.

Except for Annemarie's comment, there are other 3 ways which may improve your staining:

1) Try Biocompare.com to find the best Abs. Higher affinity of the Abs define the signal instensity;

2) 3% H2O2 solution can kill peroxidase, and 2 mM levamisole for phosphatase can decrease non-specific background;

3) Optimized buffer and program for antigen retrieval can also make a difference.

In addition to direct conjugation as mentioned by Annemarie, you can try to get very specific with your secondary antibody. If your primary monoclonal rabbit antibody is a certain subtype of IgG (e.g. IgG1, IgG2a, etc.) or has a known light chain (lambda or kappa), try finding a secondary antibody specific to that subtype or light chain that has also been cross-adsorbed to other interfering rabbit antibodies. It might not reduce all background, but it should certainly help.

Thank you very much Annemarie Honegger , Shiqiu Xiong and Maureen Rebecca Newman I would definitely try every suggestion here. I am hoping it would reduce atleast some background staining.

in addition to using a direct conjugate, you need to block FC receptors, you can do this by pre-staining the tissue with normal rabbit serum.

If you have an issue with endogenous peroxidases (like MPO) in macs or neutrophils, there are reagents to block that signal so you only read HRP signal

Immunohistochemistry on the mouse tissue utilizing mouse monoclonal antibiotics presents a challenge.

Secondary antibodies directed against the mouse monoclonal primary antibody of interest will also detect endogenous mouse immunoglobulin in the tissue.

This can lead to significant spurious staining ,therefore ,"mouse -on-mouse "staining strategy is needed to yield credible data.

This paper presents a method that is easy to use and highly flexible to accommodate both an avidin -biotin detection system as well as a biotin detection system as well as a biotin -free polymer detection system .the mouse primary antibody is first combined with an Fab fragment of an antibody in a tube and allowed sufficient time to form an antibody complex ,any non-complexed secondary antibody complex .Any non-complexed secondary antibody complex.Any non-complexed secondary antibody is bound up with mouse serum.the mixture is then applied to the tissue .the flexibility of this method is comfirmed with the use of different antimouse antibodies reagents.these techniques can (IHC),immunofloresence (lF).

This method has also been adapted to other models ,such as using human antibodies on human antibodies on human tissue and using multiple rabbit antibodies in dual immunofloresence .

Keywords:mouse -on -mouse ,endogenous immunoglobulin ,immunohistochemistry dual immunoflouresence ,fab fragment ,multiple immunostaining

Clinical importance of

rabbit monoclonal antibody on rabbit tissues with minimal or background staining.

(1)mouse models are important tools for studying human disease and immunochemistry is often required to analyse these samples .

However ,many of the antibodies that can detect mouse proteins are also created in the mouse.

Secondary antibodies directed against mouse monoclonal antibodies of interest will also detect endogenous mouse immunoglobulin in the tissue ,leading to significant spurious staining strategies are needed to yield credible data .

(2)the goal of this was to develop a flexible working system for the use of mouse antibodies on mouse tissue ,while also creating a system for using multiple antibiotics from the same species for dual staining procedures .

The method presented in this paper uses a procedure yo create a complex of the secondary reagent with the primary antibody prior to applying the tissue using commercially available components ,.Normal mouse serum is then included to bind up any non-complexed secondary antibody .A similar strategy has been used in the past ,this method more flexible than commercial kits ,because it can be used with standard avidin/biotin reagents as well as biotin free polymers .in addition ,this staining approach has easily adapted from histochemical detection systems to flouresence staining ,and further adapted for its use in multi -colored immunofloresence staining.

Materials$ Methods

(1)Primary mouse antibodies

Anti-smooth muscle action (SMA clone /A4 ,llm0851,Dako,glow,syrup,Denmark)was used at a dilution of 1:25(2-8ug/ml)after mat induced epitope retrieval (HIER) with Tris -EDTA buffet,PH 9.0(trial concentration 10mm Tris Base,lmm EDTA ) or triology (#920p,0,cell marque;Rockland ,CA).Anti-mouse actin antibody (clone HHF35 #m0635,Dako)was,used,at a dilution of 1:50 (2.12ng/ml) with no antigen retrieval etc.

(2)Basic stsining method protocol

Normal mouse tissues were fixed in formula for a minimum of the days ,and then processed and embedded and paraffin.

(3)staining method using a biotinylated goat secondary .

To create an antibody complex ,we combined a biotinylated goat anti -mouse lgG fab fragment (#115-067-003;Jackson ,immunoresearch laboratories etc.

(4)staining method using an unconjugated goat or rabbit secondary to create an antibody complex,we combined the unconjugated secondary ,either a rabbit anti mouse lgG fab fragment( #315-007-003,Jackson immuno research etc imaging.

Brightfield images were captured using a Nikon PS-Fi1 camera (Melvin,NT).Immunofloresence images were captured using the aperio F2 (vista CA),which utilities a black and white monochrome camera.Each flourochrome is represented as pseudo -color in images.

In summary

-To demonstrate the strength and pattern of staining when the secondary antibody binds to the endogenous mouse antibodies.

-once optimized ,we test this technique with a number of mouse monoclonal antibodies to verify that the staining pattern were correct for a number of antigens

@Emmanuel thank you very much for your input. I will try as you recommended.