I'm trying to perform quad-IF staining on frozen tissue sections on a fluorescent widefield (axioplan) microscope. I was wondering what people might suggest for colour/strategy for a co-IF experiment for 3 targets + DAPI? An Issue I have is that two of the primary antibodies are mouse monoclonal Igg1K and 1 of the antibodies is a mouse monoclonal IGG2a. When doing triple IF It seemed to work fairly well to do Zenon labeling of 647 and a 488 anti-mouse IgG antibody but i'm not sure best strategy for four color. I could try to find a non-mouse antibody for one of the Igg1ks and then use highly cross-adsorbed secondaries but not sure which color combination would be best.
Any help on strategies would be greatly appreciated. Thank you!
For the reliable data, you should have all targets on the different species with the excitation-emission channels well separated, i.e., DAPI, rat Alexa Flour 488 nm, rabbit Alexa Flour 595 nm, mouse Cy5 647 nm. Otherwise, you need to demonstrate the specificity of your secondary antibodies binding separately to IGG2a and Igg1k. In terms of false colors, I usually use RGB + white or RGB + Cyan; DAPI is traditionally blue, the rest is purely how well it represents the findings. In your publication, you will still need to demonstrate each channel separately and the merged data.
I've used DAPI, Opal520, Cy3 and Cy5 : this gives no bleedthrough between channels (and is very pretty).
https://wellcomeopenresearch.org/articles/5-76/v2
Any analogous fluorophores would work (alexa 488 instead of opal 520, alexa 546 instead of Cy3, etc).
I've also tried DAPI, Alexa488, Alexa594 and Alexa647: this is more fiddly, and 594 and 647 seem to cross-talk a bit, especially if you use a broad texas red TX2 filter (I use an orange filter to pick up the 594 so there's no 647 in it, and then subtract this from the far-red 647 image to clear up the cross-talk).
Hey Samuel,
This depends on the filter sets of the microscope you are using. Best check before with single stainings, if there is a spill-over between channels.
For 4-colour IF I use either DAPI/ AF488/ AF555/ AF649 or DAPI/ AF488/ AF594 / CF750, because I had signal from 594 in 649.
CF750 works very nicely, but unfortunately few microscopes are equipped with a suitable filter.
Best,
Theresa
Thank you. John Hildyard The Red Filter I'd be using would be this: https://www.micro-shop.zeiss.com/en/us/shop/filterAssistant/filtersets/489064-0000-000?pid=null&oid=object-0000-029&ff=1
I know the microscope also has a YFP filter but not sure the emission spectrum. Between 568 and 594 and 546 would you likely choose 568 to pair with 647 and 488? It sounds like 594 may have bleedthrough.