I have run into a strange problem: my immunofluorescence stainings, done on PFA-fixed paraffin sections using an Alexa546 labelled secondary antibody and mounted using Dako Immunofluorescence mounting medium, fade out in about 5 seconds when viewed in a normal epifluorescence microscope. I have used these reagents for years and never had such a problem. I have changed nothing in the protocol, and the other antibody (Alexa488) on the same section is fine.
What on earth could cause such a behaviour?
This is quite a standard immunofluorescence staining, except that I'm using 0.1% Sudan Black in 70% ethanol after the secondary ab to get rid of the autofluorescence induced by the PFA fixation. I have always done this and it has not caused any problems. The lot of the secondary antibody is quite old (expired quite some time ago) but I can't see how that could cause this kind of behaviour (the staining is perfect at first, but fades almost immediately).
Hi, Mikael, it is really strange.. Does same happens with the other set of Alexa antibody (Alexa 555, SAlexa 647)? It looks like that the problem is in antifage reagent: they do not scavenig ROS any more.. I would suggest to prepare new stock of Sudan Black and wash samples from ethanol. May be ethanol interact with antifage reagents and elimainte antifage properties. Good luck!
Thanks for the comment! I'm currently using only Alexa488 and Alexa546, and the 488 on the same double stained samples is fine (as has been the 546 previously!). The ethanol is always very carefully washed away in running water.
Since you mention that your lot is quite old, I would say it is a good idea to try another (newer) batch of the Alexa546 labelled antibody. Or did you already do this?
Hi Mikarl,
Try not washing in running water. Buffers are recommanded. Also controll ph befor applying antibodies. There are often surprises with running water. Y. S.
even without anti-fading it should not bleach that fast
since you have used this procedure before with the same equipment ?
makes it even more puzzling. All I can say is that it looks like what I've
experienced using R-PE on a CLSM (5 seconds or less, but in this case it's a flow cytometry dye used in a confocal based laser system)
Hi Mikael,
I have had the same issue in the past and a good fiend of mine from LifeTech told me that AlexaFluor546 even though outperform Cy3 in photobleaching its behaviour is quite poor. Also, antifade reagents, which I assume you are using, offer little (if any) protective effect for AlexaFluor546 dye particularly (in any of its conjugated forms). This is completely different to what happens with most other AlexaFluor dyes. So, what was the solution he gave me? well, since photobleaching was the problem then he suggested to use AlexaFluor555 which is spectrally similar to AF546 BUT is much more photostable...guaranteed. That's what I did and I never used AF546 again ;-)
Good luck!
Wow, thanks for the plentiful comments! I'm beginning to believe that this indeed is because the lot of the secondary ab is too old (it seems we actually have two lots in the freezer, and this time we probably used the older one). Quite surprising though that it would manifest like this.
I didn't actually know that Alexa546 is so susceptible to bleaching, have been thinking that this shouldn't be a problem with any of the Alexa dyes. So thanks for the excellent tip Luciano, I think I'll have to order a new secondary anyway and I will probably get the Alexa555 this time!
Regarding the tap water wash, you're right that it sounds like a risk to me. But in my experience the antibodies tolerate that surprisingly well. I have sometimes accidentally washed the immunofluorescence stained slides in HOT tap water (because that's what I usually do for pure hematoxylin-eosin stains). I thought I destroyed the staining, but in fact it was perfectly all right! Of course not something to recommend though.
Hi Mikael,
I agree with Luciano Martelotto: used AlexaFluor546 in the past and also had fading issues (nothing as acute as you described but still significant), so i switched to AlexaFluor568 and never had a problem again. I would change the secondary antibody.
I agree with Luciano, in my hand Alexa546 is less stable to compare with Alexa 555. However, in my hand I never observed bleaching in 5 seconds, even without antifage. So, perhaps, some other factors may be the reason of so fast bleaching.. Moreover, if you plan to order new antibody, I would suggest to use Dy-light which have a lot of advantages to compare with Alexa: photo-stability, resistance to pH, more brightness etc. More information you can find here: http://en.wikipedia.org/wiki/DyLight_Fluor
I have used DyLight for quite long time and I never have problem with it..
One can find DyLight antibody from many company for reasonable price.
The tap water might also be the problem. If the water has a high ion content you might be quenching the fluorescence rather than photobleaching. Photobleaching usually requires long time-scale and/or high energy light exposure.
Conjugate polymer can maintain a good space-time correlation for the stability of the property.
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