In memoriam of all the immunofluorescence I did during my PhD that did not work. I hope this will help someone

There are three principal problems that arise from doing immunofluorescences :

- There is no staining

- The staining is there but the background noise is terrible

- There are some staining but it is very aspecific

To understand such issues (mainly point 1 and 2), let's review how we (generally) perform immunofluorescence (IHF).

General protocol for IHF

FIRST DAY

1: Rince your brain slices in PBS-1X Three times (10 min each). This step can be skippable if your slices are already in PBS, if there are however in cryoprotectant, it is necessary to proceed this way.

2: Incubate Donkey or Horse Serum (Usually 5% in PBS-T 0.5%) for about 1 hour.

• this step is to block non-specific staining. Here's a good article that explains it in more details. https://www.abcam.com/en-us/technical-resources/guides/ihc-guide/blocking-in-ihc#:~:text=PDF-,Protein%20blocking%20in%20IHC,with%20primary%20and%20secondary%20antibodies

3: Remove the PBS-T + Donkey/Horse serum and incubate your primary antibody (usually dilution is 1/1000 in the blocking buffer) overnight at room temperature.

NEXT DAY

4: Rince your brain slices in PBS-1X Three times (10 min each).

5: Incubate your secondary antibody (Usually 1/500) for 3 hours tops.

6: Rince your slices with PBS-1X Three times (10 min each)

7: Mount the slice, wait around 12-24 hours for the mounting medium to harden and voilà.

Issue A : There is no staining

Usually when it comes to a lack of staining, the crucial steps are step 1, 3 and 5.

Issues related to step 1 : If your slices are in cryoprotectant it is absolutely necessary that you THOROUGHLY clean them with PBS. Cryprotectant is a glycerol-based solution that protect your slices from extreme temperature and allows you to keep them for years and years at -20 or colder temperatures. Since cryprotectant is very thick, not properly cleaning the slices will result in the antibodies being "trapped" by the cryoprotectant viscosity and thus no staining.

Issues related to step 3 : Since most primary antibodies solution are transparent and usually a very few amount is taken, it's very easy to think you have pipetted some antibodies solution when in fact your did not. Always double check that you indeed have a liquid in your pipet tips before applying the antibody solution.

One other issue that can arise is also the need for a smaller dilution for your primary antibody. Usually, concentrations used for IHF are based on the data-sheet provided by the compagny that sold the antibody and previous scientific publications. Sometimes it occurs that there is no publication nor any concrete information on how to proceed with IHF for an antibody. In that case, there is two options :

• The antibody was tested in immunohistochemistry but not IHF (DAB IHC, based on enzymatic reaction) : In that case the "rule of thumb" is to take the concentration used for DAB staining and multiply it by 2 (sometimes 3) in order to have a proper fluorescence staining.

Ex : Fluorescence staining of Orexin-Neurons in the LH, IHF (1/2000)

Fluorescence Staining of Orexin-Neurons in the LH, IHC DAB (1/4000)

• The antibody was never tested previously for staining, which is unusual but can happen, then the best way is to try several dilution. The "best" way to proceed is to do a 1/1000 ; 1/2000 and 1/500 dilution and see which one work best. If this doesn't work, consider trying 1/200, if it still doesn't work, you have to consider changing antibodies.

Issues related to step 5 : It is surprisngly easy to forget to add the secondary antibody while doing an IHF. Most secondary antibody (from personal experience) work really really well, so if you followed the protocol, your slices are clean, the primary antibody works fine, then retry performing the IHF and be careful when adding the secondary antibody.

Issue B : The background noise is terrible

Usually when it comes to a huge background noise, most issues are stems from stems from step 3, 4, 6.

Issues related to step 3 : One thing to potentially ameliorate background noise is to add donkey or horse serum with the primary antibody for the overnight incubation. Adding 1% of serum can help ameliorate background noise by saturating the aspecific site.

One other step to take when it comes to background noise and this step is to try to incubate for a longer time at 4°C. Incubation at cold temperature will make the binding of antibodies/antigen take more time and can ameliorate the background noise.

Issues related to step 5 and 6 : If the background noise is still bad, consider adding one extra wash of PBS for 10 minutes for these steps.

Ex : C-Fos staining (1/1000 for primary; 1/500 for secondary). Finding good c-Fos antibody is usually difficult and often the background noise make it difficult to interpret the staining

Issue C : There is some staining but it is aspecific

Usually, the only solution for this is to change antibodies. It can happen that for some reason the antibody you ordered do not work despite what the website of the vendor says. It is worth to make a small caption showing a positive control vs. the obtained staining and try to ask for a refund (sometimes it works...sometimes it doesn't).

NB : Something weird that can happen sometimes is your antibody not working for your specific breed of mice while it works for others. It's pretty unusual but it did happen to me with rats as in an antiGAD65/67 antibody (targetting GABA neurons) did not work on Wistar rats but worked on sprague dawley.

Other potential issues/questions

-I work on mice, I have an antibody against a specific protein made in mice, will it work on my slices ?

It can yes. It depends on how specific the antibody is. Mouse tyrosine hydroxylase (staining of DA neurons) usually works very well in mice.

- Some of my slices are stained properly, others are not

It's usually because there were too many slices put in one well for the primary antibody incubation.

- My staining worked but my slices have a lots of holes in them, like swiss cheese

Ex :

It is because the brain was not properly cryoprotected. Keep the brain in 30% sucrose for 48-72 hours before freezing it and slicing it.

- On frozen section, is it possible that an antibody works for DAB IHC but not for IHF ?

Difficult to say. Some antibodies work better in classic IHC (DAB) than in IHF (Ex : Orexin antibody) but they would still work in IHF by increasing the concentration. According to ThermoFischer, an antibdoy that works in IHF will work in DAB IHC.

- What is the best alternative for IHF or IHC in general if I can't have proper staining ?

It is very difficult to have proper staining in GABA neurons or Glutamate neurons by using IHF or IHC. Ex : GAD Staining in the RMTg (Perotti et al., 2005) .

GAD staining usually is very difficult to interpret. CamKII staining is the "best" way to stain for glumataergic neurons, but is difficult to set up.

In these cases, the best alternative is to use RNAscope.