Hi Daniela,

I have done nice IF stainings with Flag and various other proteins of interest using a 488 and 549 combination. With this I have no bleed through in the channals when using a short bandpass for 488 (500-550) and a long bandpass for 549.

The primary antibody that I used for flag was this one: Flag antibodies (DYKDDDDK Tag Antibody cell signaling #2368) (1:300-1:500 under my IF condition - with 3%PF and 0,1% saponin permabilization).

For the secondary antibodys I used either Dylight 488 Rb or Alexa 549 Rb - together with either Dylight 488 Mo or Dylight 549 Mo. If the Flag-construct have good expression it stains well with both the red and green staining, if it's only moderately expressed I would go for flag 488 and do the costaining with 549

 Dylight 488 Mo Donky igg        cat.no. 715-485-150

 Dylight 488 Rb Donkey igg    cat.no 711-485-152

 Dylight 549 Mo Donkey igg    cat.no 705-505-150

Good luck with your experiments

Hey Daniela,

This is what I've used for my IFs which all worked very nicely:

HA: mouse-anti-HA & anti-mouse Alexa647

Flag: rabbit-anti-Flag & anti-rabbit Alexa488

DAPI or Hoechst for DNA and sometimes even additionally Phalloidin566 to stain Actin

Only downside of this setup is that you can't see Alexa647 with your eye but only through software since it is too far red to see with eye