Polymers as Secondary Antibodies for Immunohistochemistry

Have you ever thought about using polymers for your immunohistochemistry experiments? Take this simple quiz and find out if polymers may be right for you:

1. I order biotinylated secondary antibodies for immunohistochemistry. Yes/No
2. I use avidin/biotin complexes in my staining protocol. Yes/No
3. I have to block endogenous biotin in my tissue samples to avoid heavy background staining. Yes/No
4. My immunohistochemistry protocol requires too many time-consuming steps after the primary antibody incubation. Yes/No
5. I am taking this quiz. Yes/No

If you answered “yes” to 3 or more questions, you may be suffering from biotin fatigue, a reaction to excessive avidin/biotin usage. But don’t worry, there’s something you can take for that.

Your Prescription: Polymers

Several companies now offer non-biotin based polymer solutions that can take the place of your secondary antibody and avidin/biotin steps in your staining protocol. The resulting experiment can be more efficient and less time-consuming.

Let’s take a look at a typical immunostaining protocol (both with and without biotin) and see what this entails:

With biotin:

1. Deparaffinize, rehydrate paraffin-embedded tissue sections
2. Boil slides in citrate buffer, pH 6.0
3. Permeabilize and block sections with protein block and peroxidase block
4. Rinse
5. Block endogenous biotin—15 minutes
6. Incubate in primary antibody—1 hour
7. Rinse
8. Incubate in secondary biotinylated antibody—45 minutes
9. Rinse
10. Generate avidin/biotin complex—20 minutes
11. Rinse
12. Incubate in diaminobenzidine—5 minutes
13. Counterstain
14. Dehydrate through alcohol and xylenes, coverslip

Now let’s see this same protocol after a biotin-ectomy:

1. Deparaffinize, rehydrate paraffin-embedded tissue sections
2. Boil slides in citrate buffer, pH 6.0
3. Permeabilize and block sections with protein block and peroxidase block
4. Rinse
5. Block endogenous biotin—15 minutes
6. Incubate in primary antibody—1 hour
7. Rinse
8. Incubate in secondary antibody—45 minutes
9. Rinse
10. Incubate in avidin/biotin complex—20 minutes
11. Rinse
12. Incubate with polymer—30 minutes
13. Rinse
14. Incubate in diaminobenzidine—5 minutes
15. Counterstain
16. Dehydrate through alcohol and xylenes, coverslip

You can see by eliminating steps 5 and 8-11 and adding step 12, you can save more than an hour of your time by reducing the number of incubations and rinses. Your experiment may even finish in time for a late afternoon happy hour.

You can also choose from a wide variety of polymers available, ranging from the specific anti-mouse or anti-rabbit polymers, to the more universal polymers that can detect antibodies from more than one animal species. I am also happy to report that there is now an anti-human polymer that can detect human antibodies, an important tool for investigators studying antibody-based therapeutics.

Is the Prescription Working?

Now, before you completely commit to polymers in your immunohistochemistry experiments, be sure to do a side-by-side comparison with your current method to ensure that the new protocol yields acceptable results.

If you’re still feeling somewhat skeptical, check out these recent results from my lab with a universal polymer that can detect antibodies from mouse, rat, rabbit and guinea pig:

Figure 1: Cell lines were fixed in cytospin solution, centrifuged onto slides and stained for HER2. The right panel shows BT474 human breast cancer cells stained for HER2 (brown DAB reaction product), while the left panel is the isotype antibody control slide. The universal polyvalent polymer was used in place of a biotinylated secondary antibody and avidin/biotin complex.

You can see that these results are very clean, with low background and good specificity.

So, in summary, polymers are a welcome addition to the immunohistochemistry arsenal of reagents. They can be specific or broadly reactive, reduce the time of staining experiments and eliminate the need to block endogenous biotin in the tissues being examined.

Please consider it as a possible remedy the next time you are experiencing biotin fatigue.

Want to know more about histology and immunohistochemistry? Visit the Bitesize Bio Histology Hub for tips and trick for all your histology experiments.