Week 4: troubleshooting

Solve your fluorescence staining problems with these tips, covering problems such as no signal, non-specific staining, and more.

There are numerous factors that can potentially affect the outcome of your immunofluorescence (ICC) or immunohistochemistry (IHC) experiments. To help you quickly find the cause of your problem and test out solutions, we're compiled the most frequently asked questions (FAQs) and a troubleshooting guide.


1. Troubleshooting ICC and IHC guide


This detailed guide takes you through numerous causes to the problems you may be experiencing in ICC and ICC and what can be done. This includes no staining, non-specific staining, high background and using controls.

Get the guide


2. Quick video troubleshooting


If you'd like to quickly find out how to solve problems with no staining and high background, our two quick videos below highlight the major causes and what can be done to solve these problems.

No signal

High background



3. Immunohistochemistry FAQs


Below you'll find answers to the questions we get asked the most about for IHC and read further information on the topic.


Video transcript for "Troubleshooting - No Signal"

So if you have no signal, this indicates the start with the need to optimise the protocol. You might have a defective antibody or there may be a lack of target in the sample. And you can check that with a literature search to see how much target should be there. So the type of things that we would recommend are to increase the antibody concentration and to make sure you incubate overnight at 4 degrees. So increasing that concentration should mean that you increase the amount of staining there. Remember to permeabilise if it’s a cytoplasmic or nuclear protein. Don’t let the cells dry out during the antibody staining, this can give patchy staining. So some areas might have no staining, whereas other areas may have too much staining. Make sure you’ve had the correct culture conditions of the cells for expression of protein. And again you can check the literature for this. It’s always good to include a known endogenous positive control and this again will indicate if your procedure is working well. Ensure the correct secondary antibody is being used and ask yourself if it’s working well with other primary antibodies just to make sure it’s working well for you.

Video transcript for "Troubleshooting - High background"

So we move on to high background. So if you have high background it is usually from excess of antibody or autofluorescence or maybe from non-specific binding. So for this you need to do the opposite in a way you need to reduce the antibody concentration make sure you are incubating overnight at 4 degrees, insure that you are including wash steps. Wash 3 to 4 times for 5 minutes for each wash step and insure you include a mild detergent like 0.2% Tween 20 in the wash buffer and antibody dilution buffer. Again you want to check that you have the correct culture conditions and that the cells are not over confluent. Ensure that permeabilization if cytoplasmic or nuclear target that sounds quit counterintuitive but I have found from my own experience and experience from our customers that if permeabilization is not done when required perhaps for a nuclear target for example that you don’t get no stainig you actually get a lot of excess staining for some reason. So thus make sure you permeabilise if you need to. Again you could check the secondary antibody to make sure it is not binding nonspecifically by including a non-primary control. Sometimes aggregates of the secondary antibody can give a sort of speckled appearance within the sample so you can mix the antibody well before use, if you want to you can even filter the antibody solution before you use it if necessary. Some cells are quite autofluorescent you may want the literature for this so they may also be autofluorescence only at certain wavelengths so if you choose a different conjugate and view in a different wavelength you may find that you correct that problem. Some cell culture matrix components and proteins again can be quite autofluorescent and again perhaps you can change the conjugate that you are using to view it at different wavelengths.

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