Hello Nicolas,

Secondary antibodies will recognize through Fab domain the Fc domains of Ig(G,M,...). In the serum, you have Ig.

Because Fc domains might share similarities between species, you can avoid this by using a serum belonging to the same specie than the secondary antibody.

You will mask all the unspecific binding sites in the same way and with something that looks like your secondary antibody and then "normalize" your noise.

Imagine you want to recover randomly a hill with tree-seeds. Only one will give a red tree. By using the same species of tree, with the same growth rate, the same size, it will be easier to find the red one.

I think you can use the second method only if the Fc domains are different from the specie of your secondary antibody and the species of your tissue/cell you want to stain.

Sami

Hello Nicolas,

Secondary antibodies will recognize through Fab domain the Fc domains of Ig(G,M,...). In the serum, you have Ig.

Because Fc domains might share similarities between species, you can avoid this by using a serum belonging to the same specie than the secondary antibody.

You will mask all the unspecific binding sites in the same way and with something that looks like your secondary antibody and then "normalize" your noise.

Imagine you want to recover randomly a hill with tree-seeds. Only one will give a red tree. By using the same species of tree, with the same growth rate, the same size, it will be easier to find the red one.

I think you can use the second method only if the Fc domains are different from the specie of your secondary antibody and the species of your tissue/cell you want to stain.

Sami

Dear Friends

There are logical facts in biological interactions of antibodies and anti-antibodies. I agree with Sami that Secondary antibodies would recognize through Fab domain the Fc domains of Ig(G,M,...) in the serum, we have antibodies (Ig) against the target antigen in the tissue or cells under study.

In my experience, there is no need of such blocking as nonspecifically bound secondary antibodies are washed off with wash buffer containing tween-20.

CS Rayat

In answering the question what people are usually doing: in our lab we use a commercially available kit for automated staining. this kit doesn't contain a normal serum blocking step, but the recommended antibody dilution reagens contains some kind of detergens and proteins (like BSA).

in my hand I use goat serum on human intestinal tissue with quite nice results

sami

I have worked with mice as well as human tissues. I have used VECTASTAIN Universal ABC kit, which has horse serum which doesnt give background. You can also use polymer based kit (like Dako Envision Flex Kit)  which requires no serum blocking but gives results due to high sensitivity.

Hope this helps. All the best

When I was working with the same protocol from vector labs I tried both the regular serum blocking and also used BSA instead of serum. Both gave me identical results. The basic idea as you have mentioned is to reduce noise due to non-specific binding. 

In many protocol researchers block non-special antigens before applying primary antibody and in continuous it usually isn't used before secondary antibody. BSA can be used as general and goat serum as a special blocker.

Hi Nicolas, I don't see any reason to do such a thing (unless all else fails!)

We incubate our tissue in PAXD (which has 5% BSA in it, PMID: 18852455) prior to adding primary antibody, and do both primary and secondary antibody incubations as well as all the washes in PAXD as well. Our secondary antibodies are raised in goat or donkey, and I've never used serum from these animals in my stainings...

If you are having a lot of background/aspecific staining, I'd first try preabsorbing my antibodies (you can do both 1° and 2° if you're using a 2° that you're not very familiar with) in the same tissue that you are staining. That way, you should eliminate most of the background/aspecific staining.

never use a serum, this contains antibodies of unkown specificities, is anything but a standard. There is a simple solution: Gelafusal. It's a hydrolysed gelatine use in medicine as plasma expander. So you are using a drug with all the checked quality parameters you can get according to the drug laws. 

http://www.medsafe.govt.nz/profs/datasheet/g/Gelafusalinf.pdf

Interesting alternative Stephan!

The link that you had there was blocked or something by my browser so I looked up one on Vector at https://www.vectorlabs.com/data/protocols/BA-9100.L.pdf

Here the quote is " If this biotinylated antibody is to be used in tissues, which may contain cross-reacting endogenous immunoglobulins, dilution of this biotinylated antibody may be made in buffers containing 2% normal serum from the same species as the tissue."    Vector provides 2% HORSE serum as a generic blocker in PBS in the Universal kits it sells as a convenience.

What I have done for many years is use 5% nonfat dry milk in 50mM TRIS + 1.5% NaCl with 0.1% Triton X-100, pH7.4 as the dilutent  for the primary and secondary antibodies.  This was given suggestion was given to me by Dr. L. Sternberger when I was starting IHC back in 1987 and using polyclonal primaries from "neat" serum which tend to have a lot more background.   Just kept it as my blocking and diluting buffer and it works fine.  You might want to adjust the NaCl to your preference to 0.9%, I have used both and works very well in comparisons.  Works well, universal and  the non fat dry milk powder can be found at any grocery very cheaply.  

By the way:  I work with both mouse brain and human brain with various antibodies for neurological studies in dementia.  Good luck in the lab!

Thank you Rose!

interesting the blocking with nonfat milk. I will give a try once on a problematic antibody!

In general, using a normal serum with secondary antibody is not necessary.We had good results during the last two decades of the last century when working on human brain (normal and pathological). Our rule was to operate with the highest dilutions possible both for primary (polyclonal) and secondary antibodies.  This leads to a drastic reduction in background.

In rodent brains (rats, wild and transgenic mouses and gerbils), we have added both a normal serum (from donkey, goat or horse) and BSA with slight improvements.

We think that better you dilutate your antibody, more you have less background. 

I totally agree with the lowest dillution the least background.

But reducing aspecific binding of the secondary antibody can anyway help. In this focus, use of serum or of other peptides will definitely help.

Of course Nicolas. You can add normal serum and  BSA with the secondary antibody.

Thank you for your nice answer, as you said Mohamed several papers are bringing this topic, such as:

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3216515/

They said: "We determined that traditionally used protein blocking steps are unnecessary in the immunostaining of routinely fixed cell and tissue samples."

Every time someone reply this post I am thinking to this paper and it is really surprising. Really need to try it!

Normal serum this same species as secondary Ab, used before primary Ab (for example 1% goat serum 0,5h in PBS, primary rabbit in PBS, washing in PBS, secondary goat anti rabbit).

Thanks Luca!

To my experience (working with mouse brain), blood in vessels are binding anti mouse antidodies (and even anti rat) and can´t be blocked even with high amount of blocking. I did not observed problem with the rest of the brain tissue.

Hello Nicolas (and everybody),

In my opinion, this new way to use the serum of the animal in which the staining is perform maybe is due to avoid autoimmunity, I mean, the secondary antibody is made in a third animal against a second animal (against the primary antibody, which is made in a second animal to detect an antigen for the first animal), so, if you add the secondary antibody with its own serum, mayby that antibody could recognise by autoimmunity its own proteins (something unusual, but why not?). This is my explanation.

I am going to repeat this but in an example:

If you would like to stain by IHC a tissue of mouse (first animal),

1º- you use a primary antibody against mouse raised in rabbit (second animal)

2º you use a secondary antibody against rabbit´s Ab raised in goat (third animal)

Usually, you would use a serum from the third animal, in thsi case GNS (Goat Normal Serum) but in this question you are affirming that the serum used is from the first animal, mouse. In my opinion, this could be because if you use a different serum that is not from the animal in which the secondary antibody was raised, and neither from the animal that the secondary antibody recognise, and you add a serum from another animal, in this case the first animal, you could aboid autoinmunity and inespecific recognition respectively, because is much less likely that secondary antibody recognize an antigen from a third animal which it has been never in contact with.

Use serum that is from the same species as your secondary antibodies.

E.g., you use Goat anti-donkey....use goat serum. Make sure it is not the same species as the primary!

BSA works for most things, but for certain antibodies using the serum from the same species is important.