I am trying to stain Marc145 cells infected with PRRSV. For this I am using an unlabelled primary antibody against N protein of PRRSV and using Alexa 546 as secondary antibody. After staining I can see non-specific staining in non-infected cells. 5% goat serum in PBS is used as blocking buffer. Can anyone suggest me how to avoid non-specific staining?
https://www.researchgate.net/post/How_to_get_rid_of_non-specific_signals_in_the_staining_of_immunofluorescent_microscopy
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Your blocking buffer is correct I would add 0.05% tween, but it is overall optional. It appears that you're secondary interact with the target cells. You can check if it is the case by incubating a blocked sample without the primary antibody. You can try to increase blocking time (i.e. 4C overnight), decrease secondary antibody concentration double the dilution factor i.e. 1:1000 to 1:2000, increase washing (Tween 20 0.1% in PBS x3 times). If nothing else works you can try to change the secondary ab. It will also help if you can present the example of the nonspecific staining and your staining protocol. Then I can provide more advice.
Anton Lennikov Thanks much! I will let you know in case it does not work.
I accept professor Anton Lennikov’s answer. Dear Shamiq Aftab every time blocking buffer should prepare freshly. After the blocking step, you should not wash. After the primary antibody incubation to wash 5 times with TBST buffer (PBS+0.1% Tween 20). First see the primary antibody concentration recommended by the product manual, because that also one reason for non-specific binding.
Niranjan Chellathurai Vasantha thanks much. Will try and let you know.
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