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Getting the best out of your in situ hybridizations requires choosing the right protocol, deciding if sections or whole mount is better, using the right equipment, making fresh buffers, careful planning for all steps, optimizing your probe concentration, and taking the time to get the development step right.

It’s Monday morning, and you’re about to start your first in situ hybridization (ISH). You glanced at the protocol on Friday, and it seemed pretty easy. You just do a lot of washes and incubations, right?

There are lots of ways ISH can go wrong. If you’re not well prepared, you’ll most likely be tearing your hair out by lunchtime. Allow me to talk you through some key points so that you don’t end up traumatized.

7 Tips for in situ Hybridization Success

1. Choose a Valid Protocol

If you’re working on a model species, someone has likely done ISH on the same species and tissue as you. Check publications for a customized protocol and save yourself some unnecessary troubleshooting.

If you need further guidance or want to ask a specific question, you can always email the corresponding author directly for insider tips.

If you’re working on a non-model species, you might need to find a protocol for something as similar as possible.

2. To Section or Not to Section

You should consider whether performing ISH is better on sections of tissue or the whole-mount ISH (WMISH). This will very much depend on your sample.

For example, if you’re working on the ovaries of a zebrafish, you probably need sections. If you’re working on tiny sponge larvae, you might be able to just do ISH on the whole damn thing (no sectioning required!)

3. Make Sure You’ve Got the Right Equipment

To make sure you don’t lose your samples (and sanity) make sure you get slides with an adhesive coating, like Superfrost Plus coated slides, so your samples don’t slide off when you put them in solution.

Other essential items include a hydrophobic (PAP) marker pen to localize the tissue on the slide, a special dark box to keep your slides in during the protocol, and possibly some flexible coverslips to stop the liquid from evaporating off the slides when you incubate them overnight.

4. Make Fresh in situ Hybridization Buffers

It’s normal to need a bunch of chemicals and buffers, but the real kicker here is that you need (almost) all of them fresh on the day you use them. Some of them you might need to autoclave before use, or have on a magnetic stirrer for a while, so make sure you allow plenty of time for this.

Hot tip: even if you get desperate, don’t use the leftovers your lab buddy has on their bench from 6 months ago. Trust me.

Every buffer you use also needs to be RNase-free, so before you make everything else, you need to make or buy RNase-free (DEPC-treated) water first. You’ll need a lot of it, so make a big batch (~2 liters).

Make sure you have enough of each solution too. As a general rule, you’ll need somewhere between 200 µl and 500 µl per slide, per treatment, to cover all the sections in solution.

5. Think Long-Term

Your protocol may have conveniently left out the fact that it will take a minimum of three days to get through this in situ hybridization step alone.

That’s not including all the sample preparation, sectioning, and making chemicals beforehand and the color development, post-treatments, and taking photos afterward.

Start on a Monday so that you don’t end up stuck in the lab all weekend. You have been warned.

Remember that for most of these 3–5 days, you will constantly be doing 5-, 10- or 20-minute washes, so you can’t leave the lab for very long to eat or drink. Make sure you keep some water and snacks on hand.

6. Check Your Probes

Probe concentration is important! As a rule of thumb, a highly expressed gene can be detected with concentrations as low as 10–50 ng per mL of hybridization buffer.

If the gene of interest has low expression, you should up the concentration to somewhere near 500 ng/mL. If you have no idea about the relative expression, you can try 200–250 ng/mL and see what happens. If it doesn’t work, increase the concentration next time.

7. Get the in situ Hybridization Pictures

The color development step can vary a lot. It could take just 30 minutes (check regularly!), or it could take several hours. If you’ve been checking every 30 minutes for several hours and nothing happens, you might need to just cross your fingers and leave it overnight.

Generally, it’s safer to leave the samples in the fridge if that’s the case, as this will slow the process down. You don’t want the color to over-develop because everything will be a purple mess.

Once you have a good amount of signal, you need to stop the reaction, then dehydrate and mount your slides with coverslips. Take the pictures ASAP, just in case they fade, or someone accidentally throws them in the bin. I know you’re exhausted by this point, but you don’t want your week of hard work to go to waste.

ISH in Summary

I hope that these tips help you perform successful in situ hybridization. The technique is complex, and there are many reasons it might not work, especially if it’s your first time trying it—mine didn’t!

So don’t give up. Keep putting these tips into practice, and you’ll get great results.

Got any tips of your own? Let us know in the comments section.

Originally published November 2019. Revised and updated March 2023.

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  1. Before beginning this work, remember to pre-treat all the plasticware/glassware with the DEPC water overnight at least. Start with new if possible. Then keep the RNAse-free lab ware specifically for this work.

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