Question about using IBEX for immunofluorescence of adherent murine cell line on 96 well plate.

[Sai101396]

Hello, I am a second-year PhD Student at UTMB (Galveston, Texas). For most of the IBEX protocols, I see they are based on tissues. Is there a protocol for just cells? I want to try using it for immunocytochemistry. The cells (in 96 wells) have already been plated, fixed, used with primary antibodies and screened with Cell Mask Blue, 488 and 568. I want to try to use the same IBEX protocol for the cells to de-stain and re-stain. Any suggestions or feedback is greatly appreciated.

[radtkea]

Welcome to the community! We are so glad you are here and are happy to support your PhD work.

Yes, published work on IBEX has focused on tissues but there should be no reason why the method would not work on plated cells. This is a very interesting application, and I am excited for you to get it to work!

We have never used Cell Mask Blue, and therefore I am not sure if it inactivates with LiBH4. Ideally, it would be great if the signal remained, and you could use it as a fiducial for each round. I suspect that Cell Mask Red might not dye inactivate and could serve as a fiducial.

AF488 dye inactivates but AF568 does not. You can see the full list of fluorescent probes tested here. There are several alternatives to AF568 that you can try such as AF555, iF555, PE, and eF570.

In conclusion:

1. You will need directly conjugated antibodies in AF488 or AF555/PE/iF555/eF570.
2. You will need to test if Cell Mask Blue dye inactivates. If yes, then you will need a different fiducial (DAPI/Hoechst or Cell Mask Red?). If no, then you can use it as a fiducial.
3. Hopefully, fixed cells are adherent to the coverslips over multiple rounds of imaging. If not, then you might need to pre-coat the coverslips with an adhesive. TBD.

Happy to chat some more, if you would like.

Best wishes,
Andrea

[Sai101396]

Hello Andrea

Thank you so much for your response and help! So the cells are already fixed into the 96 well plate wells (no cover slips, the optical plate is good for immunocytochemistry for imaging). My first round of antibodies included Paxillin and Synaptopodin. For the fluorescence, I used Cell Mask Blue, Goat anti Rabbit 488 (IgG) for the Paxillin and Goat anti Mouse 568 IgG for Synaptopodin. So in the 96 well plate, the first time I tried it with my plate was by washing with 1X PBS, 3 times (100 uL/well). Then I made the 1 mg/mL LiBH4 (0.005g of Lithium Borohydride to 5ml Millipore water - this is how I was told in the lab to make it). I then added 80 uL of the 1 mg/mL LiBH4 per well. I then did the photobleaching with the AF488 and red channel (using the mircoscope), for each for about 5 minutes. I tried blocking and restaining with another set of antibodies, but the fluorescence is still there.

Even if the Cell Mask Blue does not inactivate, I think that should be fine, meaning the next round, I could just re-block and use another set of Abs.

So, I am in the midst of trying to plan and optimize this to see if I can get this to work for my immunofluorescence. Any suggestions/advice/feedback is greatly appreaciated! Also what is your opinion on using 1 mg/ml Sodium
borohydride (NaBH4) in PBS? Would this reagent work?

Thank you,
Sai