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Used PBS instead of PBST during blocking and primary ab treatment in Immunofluor


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#1 smlprdd



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Posted 14 August 2013 - 02:52 PM

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.

Edited by smlprdd, 14 August 2013 - 02:54 PM.

#2 Curtis


    Metaller Scientist

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Posted 14 August 2013 - 11:02 PM

I only wash with PBST after incubation with secondary antibody.

#3 Tabaluga


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Posted 18 August 2013 - 11:37 AM

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...

Il dort. Quoique le sort fût pour lui bien étrange,
Il vivait. Il mourut quand il n'eut plus son ange;
La chose simplement d'elle-même arriva,
Comme la nuit se fait lorsque le jour s'en va.


#4 bob1


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Posted 19 August 2013 - 01:25 PM

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

#5 BMF



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Posted 19 August 2013 - 02:12 PM

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.

Edited by memari, 19 August 2013 - 02:16 PM.

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