How To Test Your Antibodies (II)

Things to note when screening antibodies

1. Tissue dissection

Be sure to include the tissue region of interest known to express the antigen. For example, if the antibody targets cytokeratin and a human colon sample is used, epithelial tissues must be included during the dissection process. At times, especially for developmental markers, samples of appropriate biological age are crucial to antibody evaluation.

2. Know your antigen

A thorough literature review is key to interpreting the images of the antibody screening results. Key attributes are its antigen expression pattern, expression levels and expected intensities, expression heterogeneity, and subcellular location (nuclear, cytoplasmic, granular or membranous). 

3. Know your antibody

Ensure to read the antibody concentration from the vendor’s specifications. Using too little antibody will yield a very weak signal that is difficult to interpret. Be sure to use the antibody in microgram amounts rather than in dilution. If the antibody is in the form of a serum or ascites fluid, you can treat the total protein to have 2-5% of the desired specific antibody.

Furthermore, if the antibody is specified for a particular species (such as human only), there is a good chance that it will work only on human tissues; however, it may be worthwhile to try.

Finally, if the antibody is specified to work only in Western Blots (WB), ELISA, or immunoprecipitation (IP), but not IHC or IF, it is very likely that it will not work with 3D immunostaining. Antibodies marked as applicable for IHC in paraffin sections are very likely to work, while those applicable for IHC in frozen sections have a slightly lower chance. Antibodies marked as suitable for immunofluorescence (IF) are further less likely to succeed.

4. Choice of fluorophore

We chose CF647 dyes because they give minimal nonspecific staining, while the far-red fluorescence minimises autofluorescence interferences. Popular dyes like AlexaFluor 647 are highly negatively charged, which often leads to granular staining in positively charged regions, most notably those in eosinophils, neutrophils, and Paneth cells of the gut. 

We found that the use of CF®647 eliminates these kinds of nonspecific staining, as it is a PEGylated rather than a sulfonated dye like the AlexaFluors. This is independent of the choice of secondary whole IgGs, Fabs, and nanobodies, which will always produce nonspecific stainings when AlexaFluor 647 is used, but not with CF®647.

5. Mouse-on-mouse staining

Mouse primary antibodies applied on mouse tissues may produce nonspecific staining on macrophages, microglia, dendritic cells, granulocytes, and occasionally endothelial cells due to the expression of Fc receptors that bind to the Fc region of the IgGs. You can pre-block the tissue with mouse nonspecific IgGs to eliminate these nonspecific stainings.

6. Counterstain 

The inclusion of DAPI and additional counterstains can significantly aid in interpreting the images, especially with unfamiliar antigens where little data is available.

7. Imaging

A high-quality image is essential for the accurate interpretation of the results. Light-sheet fluorescence microscopy typically has insufficient resolution for evaluating antibody staining. We routinely perform confocal microscopy with oil-immersion objectives (40x or 63x) for antibody screening. As OmniStain reduces the staining intensity by 2-3x, the oil objective also helps collect more light to produce a bright signal; hence, be sure not to be afraid to crank up the laser during imaging. This is especially true for weakly expressed tissue antigens (a notable example being CD31). Finally, due to the limited working distance, it may take some effort to find the right region with the expected expression, but remember that this additional 5 minutes is worth the same amount of the antibody cost and future replacement antibody combined! 

8. Patience

We typically screen our antibody twice before ruling it out as failing, as this saves future experimental costs and replacement antibodies. A good, working antibody is an invaluable asset that promises numerous stable, beautiful 3D images of great utility.

Why does Illumos’ OmniStain have such a vast list of compatible antibodies?

Because OmniStain is standardised, fast and highly scalable, we typically perform 30-100 antibody screening batches using 96-well polypropylene plates, which can withstand solvent-based clearing reagents. As tissue cutting can be performed with a scalpel and forceps (compared to microtome tissue sectioning), the entire process can be completed within a week.