Hi all,
I am staining F-actin in fixed spheroids with Alexa-Fluor647 phalloidin and the staining is quite stable for a couple of weeks. Recently, I observed a massive loss of actin signal within 4 days.
My staining protocol is quite standard; fixation with 4% PFA, permeabilization with TritonX-100, blocking, and Alexa-Fluor647 (in PBS). Staining usually takes several days after fixation since I stain with other antibodies. I use the non-hardening mounting medium for my samples.
Has anybody observed a loss of phalloidin stain before? and how did you overcome it?
My guess is that, while phalloidin does have a very high affinity for f-actin, there is an off rate and over time it is simply disassociating and diffusing into the mounting medium. If you are using non-hardening mounting medium you can try to remove the coverslip by soaking it in PBS in the fridge overnight, lift it off the slide with forceps, re-stain with phalloidin, and then remount.
I agree with Christopher. I've seen the off-rate from phalloidin over time. For some unknown reason it is worse with far red and near-IR dye conjugates. Ways to slow (not stop!) the off-rate include performing a post-fixation with formaldehyde (4% for 10 minutes) after the phalloidin label, mounting in a hardening mounting medium, and keeping the samples cold after mounting.
I have experienced that issue several times, since I have many years staining F-actin with phalloidin or phallacidin. If you want to have a more stable signal, it is better to make allicuotes with enough volume for few experiments (in my case 20uL) instead of taking the entire vial. Also each time try to manipulate faster to reduce light exposure. Check expiration date, but also, if the main stock was resuspended more than one year ( some times we use from several years) that also affects a lot the instability. Other recommendation is, because this is a derivated of a toxin, when you dilute it, use just PBS, be sure about the pH, etc. Antibodies are more stable during staining than phalloidin ( mainly an old one). About the mounting media, vectashiel or Dako are good brands.
A quick addition to Xochitl's comment regarding mounting media: Vectashield is known to quench far red dyes, including AF647. I recommend instead using ProLong Glass (or one of the older ProLong products).
Thanks you all for your help I tried the restaining step, it worked but with a high background. In my case I am restricted to use the non-hardening mounting media since my samples are fixed and stained in a 96-well plate.
Any suggestions for an effective non-hardening mounting media?
Fatima Hassan I would suggest the newly-released SlowFade Glass, which does not harden yet has the same antifade protection and refractive index as ProLong Glass.
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