Used PBS instead of PBST during blocking and primary ab treatment in Immunofluor - (Aug/14/2013 )
I am doing an immunofluorescence experiment on formalin fixed tissue. My normal protocol calls for doing the blocking and antibody incubation steps with Phosphate Buffered Saline + Tween-20, but I mistakenly used regular PBS. At this point, I have carried out blocking and primary incubation with regular PBS instead of PBST; the slide has not been treated with fluorescent secondary antibodies yet, and is currently washing in PBS. How much damage have I done to my experiment? Is it possible to rescue it and get a good result, perhaps by doing an extensive wash to remove nonspecifically bound primary antibody?
Thanks for your time and assistance.
I only wash with PBST after incubation with secondary antibody.
When I did IF, I used PBS for washing; blocking solution for primary incubation; and PBS for secondary incubation... what would be the advantage of adding the detergens Tween ?
Just curious because I might try it too to improve my stainings...
Tween makes the staining more specific by increasing the stringency of the binding of the antibodies. You can also try increasing the salt concentration.
You can do anything.
For Incubation of antibody you can use any ways such as:
1-Phosphate Buffered Saline + 2.5% Skim Milk
2-Phosphate Buffered Saline + Tween-20 + 2.5% Skim Milk
3-TBS + 2.5% Skim Milk
4-TBS + Tween-20 + 2.5% Skim Milk
and for washing you can use any ways such as:
1-Phosphate Buffered Saline + Tween-20 (+ with/without higher salt concentration more than 150mM)
2-TBS + Tween-20 (+ with/without higher salt concentration more than 150mM)
I use BOLD lines.
For washing, the background depends on antibody.
For reducing background, you can wash membrane more.
I usually wash 5 times each 15min. For antibodies with higher background, I wash 10 times each 7-10 min.