4 replies to this topic

[Redha Albahrani]

Hi all,

I'm recently using IHC for my project and I want to confirm something about the washing buffer as well as the dilution buffers are used in IHC: my two questions are:

1- what is better using as an dilution buffer for primary antibody and secondary antibody, TBS or PBS?

2- can I use the dilution buffer that is different than the washing buffer? for example: can I use PBS as an dilution buffer and TBS as a washing buffer at the same time or do I have to use the same buffer for both?

please could you help me with this

Thanks,

[Curtis]

I've used both of them and I had no problem, but for IF and IHC, PBS is mostly recommended. I also read somewhere that it depends on the protein you work on too. some proteins are sensitive to TBS I think.

TBS is mostly used for Western blot.

I also recommend using the same buffer.

[Redha Albahrani]

Thanks for your kindly reply.

in fact I'm using TBS currently and I tested on the cytokeratin protein and it worked perfectly with me but when I tried it with other proteins, some of them showed expression and others not. thus, I was anxious if the problem was either from the washing and the dilution buffer. I'm using abcam's antibodies and their recommendation is TBS to reduce the high background staining. Moreover, I used to dilute both the primary and the secondary antibody with TBS + 1% of BSA does that have any sort of effect on the affinity of the both antibodies. (I'm using ABC kit, goat serum for the blocking step) (the tissues i'm testing are Humans) (the antigen retrieval i'm using also proteinase K from Dako) (the secondary antibody goat anti-rabbit conjugated) and also most of the antibodies I'm using are rabbit polyclonal.

One last thing: can I add Tween-20 with PBS instead of the Triton X-100 in order to reduce the surface tension of my buffer.

Thanks I'm just glad that I could find someone to reply on my questions so please dont mind me if I'm asking many questions

[Chelo]

Hi Redha,

I agree with Curtis. In principle, you could use both buffers. However, PBS is more general and widely used.
And definitively, you can use Tween-20 rather than Triton.

[Redha Albahrani]

Thanks Curtis and Chelo for your kindly reply