How To Test Your Antibodies (I)

Omnistain is compatible with a wide range of antibodies (https://illumos.co/insihgt-compatible-antibody-list/). However, as some antibodies prefer to work in one condition rather than the other, it is important to test whether your antibody works on its own first on a trial sample.

What to expect when using an antibody with OmniStain

OmniStain greatly increases staining penetration depths (by 10-20x) while slightly reducing the immunostaining signal intensity (by 2-3x), especially when the tissue is large and only a small amount of antibody is used – the antibody just got “spread thin” across the layers of tissues throughout the volume. This is most notable for the Cluster of Differentiation (CD) antigens, especially CD31 and CD34.

OmniStain temporarily inhibits antibody-antigen interactions and reactivates them later. This ensures the antibodies can penetrate deeply and uniformly across the tissue. The process does not denature the antibodies. 

How to test your antibodies

At Illumos, we screen all antibodies using the following method. 

We first sample a small volume (~1 mm³) of reference tissue expected to express the target antigen. For example, a human spleen for anti-CD3E antibody testing. 

Divide the tissue into two parts. 

For the first part, stain the tissue following the OmniStain protocol with 1 μg primary antibody and 1 μg secondary antibody Fab fragment conjugated to a far-red dye (such as CF®647). A counterstain, such as a lectin and DAPI pre-complexed with OmniStain buffer C, will be useful.

For the second part, stain the tissue as above, but replace all OmniStain buffer A’s and buffer B’s with PBSN. This is to assess whether the compound (especially in buffer A) will affect antibody-antigen binding specificity. 

Image the stained and cleared tissues using a high magnification objective (such as an oil-immersion objective) using a confocal microscope. The clarity of the images is crucial for interpreting staining quality, especially for uncommon, weakly expressed antigens.

Compare the staining results with the literature. A helpful reference is the Human Protein Atlas (https://www.proteinatlas.org/), where we can search for our target antigen and view the expected staining pattern in the tissue type we are using. The Human Protein Atlas even has cancer tissues stained for reference.

If the staining appears satisfactory for both the PBSN and OmniStain, that means you have identified a suitable antibody. Note it down! Approximately 90% of the antibodies that work with PBSN will also work with OmniStain.

If the staining appears satisfactory only with the PBSN group, it means that OmniStain causes some interference with antibody-antigen binding. In this case, the antibody is working but is incompatible with OmniStain. We would appreciate it if you let us know.

If the staining is unsatisfactory in both cases, it means either the antibody is not working or the sample is of low quality. You can confirm this using a freshly fixed tissue. 

What antibodies are incompatible with OmniStain?

We have yet to identify the group of antigens or antibody characteristics that lead to failures with OmniStain. In most cases, we can get a good antigen staining with an antibody from one vendor but not the other. In contrast, we have yet to find an antigen where OmniStain consistently fail while PBSN is successful. These results suggest it is the quality of the antibody that matters, not the buffer conditions in OmniStain.