Written by
The MI Team
How to Optimize HCR™ HiFi Encoding for Brighter HCR™ Gold IF Signal
If you're seeing weaker than expected signal in your HCR™ Gold IF experiment, your tissue or primary antibody is usually not the problem. More often, the issue is that enough of your primary antibodies have been labeled by the HCR™ HiFi Encoder.
When this happens, unencoded primary antibodies can still bind to the target, but they cannot trigger HCR™ Gold amplification. They may also occupy binding sites that encoded antibodies would otherwise use. This leaves fewer encoded antibodies available to generate signal, which can make the resulting HCR™ Gold IF signal weaker.
The good news is that there are two simple strategies that can help improve your signal:
- Reduce the amount of primary antibody so the available Encoder can label more of it.
- Increase the amount of Encoder A/B so more primary antibodies get labeled.
What approach should you try first?
Both strategies work by increasing the fraction of primary antibodies that become encoded. The best place to start depends on how you optimized the antibody before running HCR™ Gold IF.
- If you've previously used traditional IF:
- Start by reducing your primary antibody concentration. Primary antibodies optimized for secondary antibodies are often used at higher concentrations than HCR™ Gold IF needs. Using less primary antibody gives the Encoder a better chance to label a larger fraction of the antibodies present. This can improve signal and may also reduce background. Though, if you dilute too far, too few antibodies will bind the target, and signal will begin to decrease.
- If you've previously used TSA or other amplified workflows:
- Start by increasing the amount of Encoder A/B. This lets you keep your primary antibody conditions the same while increasing the number of antibodies that become encoded. The tradeoff is that this approach uses more Encoder A/B reagents.
Strategy 1: Reduce the primary antibody concentration
This approach can feel counterintuitive if you're used to conventional IF, where more primary antibody often means more signal. HCR™ Gold IF works differently, as only encoded antibodies can trigger amplification.
In short: less primary antibody can make encoding more efficient, which can produce brighter HCR™ Gold IF signal.
Instead of trying to bind as much primary antibody as possible, the goal is to have enough antibody bound to the sample and to make sure a large fraction of those antibodies are encoded. When you dilute the primary antibody, slightly fewer antibodies may bind, but more of the bound antibodies may carry Encoder A/B and trigger HCR™ Gold amplification. Since HCR™ Gold IF is roughly ~20𝗑 brighter than direct-labeled secondaries, diluting the primary a few-fold can still generate strong signal.
Example: Titrating a primary antibody with unknown concentration
Let's say you have a primary antibody with an unknown concentration. You've been using it at a 1:100 dilution as optimized for direct-labeled secondaries. In your HCR™ Gold IF experiment, the signal is weak.
Try three dilutions side by side:
- 1:100 (your current condition)
- 1:200
- 1:800
Run all three conditions in parallel using the standard encoding protocol. Then, compare the images:
- Does the signal improve at 1:200?
- Is it similar to 1:100?
- Does it start to decrease at 1:800?
If 1:200 gives the best result, fine-tune with intermediate dilutions such as 1:300 or 1:400. If the signal keeps improving as you dilute, your starting concentration was probably higher than needed for efficient encoding.
Reducing the primary antibody concentration is often the simplest first step. If you prefer to keep the primary antibody dilution unchanged, try increasing Encoder A/B volumes instead.
Strategy 2: Increase the amount of Encoder A/B
You can also improve encoding by adding more Encoder A/B reagents while keeping the primary antibody dilution fixed. This approach is useful if you've already optimized your primary antibody and would prefer not to adjust it.
Example: Titrating Encoder A/B at a fixed primary dilution
Let's say your primary antibody dilution is fixed. Your HCR™ Gold IF experiment generates weak signal, but you want to keep the primary antibody condition the same.
Repeat the experiment with these encoding conditions:
- Standard Encoder (1𝗑): 1 µL EncoderA + 1 µL Encoder B per 100 µL Antibody Probe Solution
- 2𝗑 Encoder: 2 µL Encoder A + 2 µL Encoder B per 100 µL Antibody Probe Solution
- 4𝗑 Encoder: 4 µL Encoder A + 4 µL Encoder B per 100 µL Antibody Probe Solution
Run all three conditions in parallel. Then, compare the images:
- Does the signal improve from 1𝗑 to 2𝗑 Encoder?
- Does it improve further at 4𝗑?
- Does more Encoder increase background?
If signal improves from 1𝗑 to 2𝗑 but not from 2𝗑 to 4𝗑, you have likely reached a useful range. Fine-tune by testing intermediate amounts, such as 1.5𝗑 to 2.5𝗑.
If signal continues to improve at 4𝗑 without added background, the original encoding reaction likely did not include enough Encoder A/B. You may benefit from testing slightly higher Encoder amounts.
Final notes
Change one variable at a time. Small, focused titrations make it easier to see which condition improves encoding for your antibody.
Every antibody behaves a little differently, so don't be discouraged if your first HCR™ Gold IF experiment isn't perfect. A few targeted adjustments can make a meaningful difference. Once you find the right balance, you can use those conditions with more confidence in future experiments.
We hope these strategies help you optimize your assay with confidence!