Fast Blue Signal Showing up in Multiple Channels

Experimental Techniques
1

I injected Fast Blue into some mice and recovered the DRG.
There is clear FB signal. I am using a Leica epifluorescence microscope with an LED light source and various filters.

What I’m noticing is that I am seeing FB signal in the DAPI range using violet illumination and blue emission filter, but also using blue illumination and the green (L5) emission filter set. So both channels.

Many studies have combined Fast Blue with a green fluorophore, so I’m wondering what’s happening here.

There is no published spectrum for FB so that really limits the detective work that can be done, but I suspect that the excitation and emission spectrum for FB must be broader than just UV-Blue range. I see papers using Confocal, which will have more narrow excitation (405, 488 nm lasers) and emission filters.

Anyone ever seen this and have any thoughts?

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@tberta @thicunha @liz @sshiers @ShanTan @AlexGA @AubinMoutal @defaria

2

Hi @achamess, I haven’t used FB yet, only viral tracing but I’m not surprised with high blue signal bleeding through into green. In my experience, depending on the filter set it can be very annoying having green and blue together. Sorry for not being of help more on this topic.

3

Thanks @defaria . Yeah it’s frustrating but we’ll figure it out.

Out of curiosity, what kinds of viruses are you using to label (DRGs?). I’ve been on the hunt for something that works well retrogradely in adult mice for a long time. Haven’t found it yet.

4

I totally miss this, sorry. The one that worked best was an AAV-PHP.S-tdtomato from addgene :wink:

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