[Histonet] Help needed!!! Immunofluorescence double staining ofwith mouse lung

[Chengming Wang]

Dear ALL,

This is Chengming from Auburn University, and this is my first time
here, and I need your help with my frustrating double staining of
immunofluorescence with mouse lung.

This is cryosection of mouse lung, only fixed with 5-minute acetone
without involvement of formalin or paraffin.  I am trying to stain two
targets with Alexa Fluro 488 and Alexa Fluro 594, and counter stain with
DAPI.

The individual staining works fine. However, I found that: when I
stained with 2nd antibody of Alexa 594, I could see some signals in FITC
channel (Alex 488). And this signal from Alexa 594 to 488 really bothers
me when we performed double staining.

I tried that the autofluorescence is very very weak in all cases.  In
reading the distributions of different fluorescence dyes, there should
not be any signal spillovers from Alex 594 to 488.

Any help or suggestions are highly appreciated!!! If you have
experience with this, please let me know!

Thanks in advance!

Chengming Wang
Auburn University