I have experienced with fluorescent dirtiness/dustiness at the end of IF staining (free floating) protocols performed on frozen brain sections. I have tried to tweak secondary antibody dilution 1:1000 to 1:500 but didn't solve the problem. An example image is below.
Any suggestion is welcome.
Hello İrem Akçalı
You need to reduce the background staining. Washing is an important step in the immunofluorescence staining protocol. It’s easy to shorten this step when in a rush, but this may fail to effectively remove antibody that is not specifically bound to the target, which can lead to poor signal: background ratio.
When you wash after each application of antibody or other fluorescent probe, it will eliminate antibodies with lower binding affinity present in the sample and thus reduce non-specific signal, or cross-reactivity. Washing for a few minutes in PBS with at least two buffer exchanges will help to eliminate unbound and loosely bound antibody from the sample. Longer washes may not confer noticeable background-reduction benefit but are generally not harmful so long as extended time in buffer does not detach the sample from the slide or the coverslip.
Another aspect you need to consider is blocking. To reduce non-specific antibody binding and, in consequence, reduce background signal, it is recommended to use a blocking solution prior to incubation with antibodies. The most effective blocking solution will be that containing serum from the same species in which the secondary antibody was raised. But you should try using bovine serum albumin (BSA) at a 5% dilution which will work as a general protein blocker that can be used with any secondary antibody.
So, following proper washing steps as well as blocking may help solve your problem.
Best.
Further, a brief but high speed centrifugation of all the compounds before use is helpful to get rid of larger aggregates, esp. important before using the antibodies.
Dear Malcolm Nobre we apply blocking both before primary antibody incubation and before secondary antibody incubation. Should I try using BSA for both or only blocking before secondary antibody?
İrem Akçalı Did you also try an Autofluorescence Quencher such as TrueBlack? It will make your staining look more specific by eliminating the lysosomal autofluorescence.
Paul Scheunemann never tried it actually. Could you give me brief information please?
İrem Akçalı It´s a reagent provided by different companies such as Biotium. You can apply it pre or post-IF staining (it is comparable to older Sudan Black B) and will make your section appear macroscopically black while reducing the unspecific autofluorescence, which can be due to accumulation of autofluorescent lipids or protein in the lysosomal compartment.
Bin Zeng Thanks for the information. I am willing to know what part of the staining protocol is approriate to implement this 3x10' washing in, before or after blocking?
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