FAQ on Antibodies (part I)

We often encounter questions on the choice of antibodies, so we have compiled our insights to share with everyone interested in appreciating the beauty of 3D imaging. Selecting the right antibodies is crucial for achieving accurate and reliable results in your research. In this post, we will delve into the key considerations and tips to help you navigate the selection process. Feel free to drop us an email to inquire or share your insights!

Antibodies in the context of immunohistochemistry

Antibodies used in immunostaining usually refer to immunoglobulin (IgG) molecules, although occasionally IgM variants can be used.

Structure of antibodies

IgG antibodies are classically Y-shaped, the stem of the Y is referred to as the Fc region, while the two arms are the Fab region responsible for binding to specific antigens. 

Vertical splitting of the Y-shaped antibody

The antibody can be splitted along the symmetric middle axis of the Y-shaped molecule by reducing the disulfide bonds involved in the connection. This usually causes the destruction of Fab region as it contains disulfide bonds as well, resulting in the elution of the antibody as it loses its antigen-binding function. 
Disulfide bond reduction is usually achieved with reagents like sodium sulphite, 2-mercaptoethanol, or tris(carboxyethyl)phosphine.

Horizontal splitting of the Y-shaped antibody

The antibody can also be split along the bifurcation of the Y-shaped molecule; the Fc region becomes an Fc fragment, and the two Fab regions become two Fab fragments. The Fab fragment retains the ability to bind to antigens. 

Antibody fragmentation into Fab and Fc fragments is usually achieved with papain protease digestion. Jackson Immunoresearch has great articles and animations on Fab fragments.

Does INSIHGT / OmniStain need primary conjugated antibodies?

No. Because INSIHGT uses monovalent Fab fragments, off-the-shelf primary antibodies (as IgG molecules) can be co-incubated with fluorophore-conjugated Fab fragments, and they will not clump together. 

We recommend fluorophore-conjugated secondary Fab fragments from Jackson Immunoresearch (https://www.jacksonimmuno.com/technical/products/groups/fab). 

What are the advantages of Fab fragments?

Fab fragment is monovalent, meaning it binds 1 antigen per 1 Fab molecule. Meanwhile,  IgG is bivalent and binds two antigens. If the antigen has two binding sites (such as a primary antibody), then it will be clumped together by IgGs, but not by Fab fragments. 

Primary antibodies can be added together with secondary Fab fragments, achieving one-step indirect immunofluorescence. In contrast, bivalent IgGs will clump the primary antibodies, they must be applied to the tissue separately with adequate washings in between. This eliminates two experimental steps and usually shortens 3D immunostaining time by half.

How many Fab fragments will bind to each primary antibody?

On average 3.

Note that Fab fragments are polyclonal, hence they bind to random sites on each primary antibody IgG molecule.

Can other methods use Fab fragments to simplify immunostaining too?

Theoretically yes, as Fab fragments will not cause clumping. However, without the use of OmniStain buffer A, Fab fragments will pre-complex with the primary antibody IgG molecules. This results in a complex size of ~300 kDa, which needs 41% more time to diffuse the same distance as separate IgG molecules, which are 150 kDa in size.

For example, with iDISCO+, a whole adult mouse brain staining will take 7 days of primary antibody staining and 7 days of secondary antibody staining, making 14 days of staining in total. With Fab fragments, you can apply primary and secondary Fab fragments together, but need 7 x 1.41 ~ 10 days of staining, probably with more severe penetration non-uniformity.

In contrast, with OmniStain buffer A, primary IgG and secondary Fab fragments will remain separated, and delayed until the next incubation in OmniStain buffer B, the maximal molecular size to penetrate the tissue remains at 150 kDa. And the special chemistry of OmniStain allows shortening of immunostaining duration to 3 days, with even better penetration uniformity.