In situ immunostaining protocols

I want to image live cell behavior then fix and stain for protein structure and localization without removing the sample from the microscope, so that I can relate cell behavior and staining on the single cell level. I have never done in situ immunostaining and would appreciate people sharing their protocols. My dream is to build out an awesome automated apparatus like in NanoJ-Fluidics ( . But I would like to learn how to crawl before I try to run. I am using common mammalian MEF, U20S, and HT1080 cultured cells. Thanks!


I don’t have an in situ immunostaining protocol, but a simple alternative might be to grow your cells in a dish with a gridded coverslip. These have grids and labels that are visible in the image, allowing you to identify the same field of view/cell before and after staining. Might be worth a try before tackling staining on the microscope!


My graduate mentor did something similar in the following paper when studying motor proteins associated with formerly-moving vesicles:
Stable Kinesin and Dynein Assemblies Drive the Axonal Transport of Mammalian Prion Protein Vesicles

When I was in the lab, I helped make a JoVE article demonstrating the technique:

Our protocol is very manual and uses landmarks within the microfluidic device to help return to the previous location, so no LEGO bricks were harmed in the making of our video! Hope it helps give some perspective.

Anna’s idea to use gridded coverslips is also a good one!

If you’re considering commercial solutions, the CellASIC ONIX system is an option.

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Grid cover slips are a great way to go.
A very simple test you can do to see if you like this approach is to just scratch a slide, study a few cells near the scratch and then fix&stain as normal. Then returning to the scratch and finding the same cells shold be simple.
More limited (you have to study cells near identifiabe scratch compared to anywhere) but a simple test you can do soon.


EMD has a protocol document “Demonstration of long-term culture and in-plate staining protocols using the CellASIC® ONIX Microfluidic platform” on its website.,MM_NF-C117908#anchor_TI

Also note that the documentation for the plates say:

The plate is incompatible with acetic acid and organic solvents such as
acetone, ethanol, and methanol. Plates should be tested for compatibility
with other acids or organic solvents prior to use.

The protocol using the Cellasic ONIX via the document I linked above uses paraformaldehyde:

@jennifer, has this device worked well in a core facility setting? I was an early user via the Süel Lab now at UCSD during graduate school, but have had a harder time getting access during my postdoctoral studies.

Check out some of our publications where we do exactly this:

Correlative live-cell and superresolution microscopy reveals cargo transport dynamics at microtubule intersections, Š Bálint, IV Vilanova, ÁS Álvarez, M Lakadamyali, Proceedings of the National Academy of Sciences 110 (9), 3375-3380

Cross-talk-free multi-color STORM imaging using a single fluorophore, J Tam, GA Cordier, JS Borbely, ÁS Álvarez, M Lakadamyali, PloS one 9 (7), e101772


Yes, the ONIX has mostly worked well in our core. Getting a strong seal is sometimes a pain, but we have an old version so that may have improved. I like this design as a solution for perfusion in a core because there is no tubing to clean out.