Tips for Immuno-fluorescence
For this week, I wanted to focus on immunofluorescence (IF). Before we get into it, I wanted to tell you guys a story.
When I first started my PhD I didn’t have a lot of experience with IF, but was super enthusiastic for it. On my first attempt of imaging my protein of interest, SOX9, I was able to get beautiful intricate stainings of string-like structures. I rushed back to lab with a hypothesis that SOX9, a protein that is supposed to be in the nucleus, was miraculously co-localising with my cell’s cytoskeleton- truly revolutionary. Thankfully, a postdoc in my lab was able to see the pictures over my shoulder and corrected me- what I was seeing was, in fact, not a theory shattering SOX9 localisation, but rather just a lot of unspecific staining. Frustratingly, it took me over a month after that first initial experiment to get a specific staining, and let me tell you- the real deal looked a lot less impressive than the first unspecific staining. I’ve attached a picture below for reference.
What I aim to say with this is that IF is a very fiddly protocol, not because it is difficult to stain “something”, but rather because it is difficult to get a clear and specific staining of your protein of interest. So, let’s get into it.
Invest in time in picking out the best antibody for you.
Commercially store-bought antibodies frequently have their destined applications listed below them; Western blotting, IF, IHC, CHIP, the options are endless. Although some antibodies shall work for applications not listed in its catalogue sheet, it is not a guarantee, so you want to narrow down your possible antibodies that specifically state your future use for them. Furthermore, it is important to check out previous papers that have sourced the antibody you are considering purchasing. What you need to look for is the use of similar cells to yours. Proteins may have different localisation and different concentrations in different cells, so the key is to have a pretty good understanding of what your IF images are supposed to look like before you make the purchase and start with the staining.
Figure out the best permeabilization solution for your target of interest:
Having figured out where your protein is supposed to be located, you can start picking out the permeabilisation solution. The permeabilisation step essentially pokes holes within your cell membrane so that the antibody can penetrate inside the cell and bind to its specific target. However, this step can vary greatly; if your target is located on the cell membrane you shall want a gentler permeabilisation step, rather than if it has a nuclear localisation- in which case you shall want to increase the level of permeabilisation. The most popular detergents are Triton X-100 (ThermoFisher Cat No. 85111), or NP-40 ( ThermoFisher Cat No. 85124). I personally prefer to go with NP-40 at a concentration of 0.5% for 3 minutes, as I find that it works for both cytoplasmic and nuclear proteins, and does its job in a considerably shorter amount of time.
Primary Antibody Staining:
My main tip for this step is more of a time saving one. Frequently, protocols shall say that, as with Western blotting, it is necessary to leave your staining of the primary antibody overnight at 4°C. However, I found that if the antibody concentration is correct, it makes very little difference in the result whether the primary antibody is left overnight at 4°C or for 1 hour at room temperature. So, my advice is to first experiment with different primary antibody concentrations, staining overnight in cold conditions. Once you have determined the antibody concentration that works best for you, try and stain it at room temperature, expediting the process a little bit!
Drop the nail polish and pick up some Mowiol!
A true-life saver I have been told about during my PhD is Mowiol (Merk, Cat no. 81381), a solidifying mounting media. Previously, I would consistently struggle to fix my slides with nail polish on top of slippery mounting media and would frequently end up with an extremely messy slide and an improper seal. However, with Mowiol, fixing my slides has honestly become so easy. Yes, it is a pain to make and takes an extremely long time to dissolve. But after you have made a stock to last you years, the final step is as easy as pipetting 5μL onto your glass slide, pressing your cover slip cell face down onto the Mowiol, and letting it solidify in the dark overnight. Then, when you go to image, just wipe off the excess with ethanol, and you’re ready to image!
That’s all for now, I hope this little fly-by IF tips has been of help, and until next week!
Reagents mentioned:
Triton X-100, Thermofisher, Catalogue Number: 85111
https://www.thermofisher.com/order/catalog/product/85111
NP-40, Therofisher, Cat No. 85124, https://www.thermofisher.com/order/catalog/product/85124#/85124
Mowiol, Merk, Cat No. 81381,