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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.

-smlprdd-

I only wash with PBST after incubation with secondary antibody.

-Curtis-

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...

-Tabaluga-

Tween makes the staining more specific by increasing the stringency of the binding of the antibodies.  You can also try increasing the salt concentration. 

-bob1-

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.

-memari-