[Histonet] My frustrating experience with double fluorescence staining!

[Chengming Wang]

Dear All,

I am new with histology, and  posted yesterday a message of: Help
needed!!!  Immunofluorescence double staining with mouse lung.  Thanks a
lot for the several helpful responses.  As asked, I am here putting on
my concise protocols for your check:

Sampling:  Cut a small cut along the mouse trachea, injected neg 50
(similar to OCT). Then quick freeze with liquid nitrogen, and the stored
@ -80; After section with 6 um, slides were fixed with  icy acetone for
5 minutes. Quick dry, and then stored @ -20 till use.

Staining: 1. Washing 3 X 5 mins; Blocking Buffer (10% Rabbit serum; 5%
BSA; 0.1% Tween 20) for 1 hour at room temperature;
                2. Tapping away the blocking buffer, and applied first
antibody for 1 hour; then wash 3 x 5 min'
                3. 2nd antibody 1 hour; washing 3 x 5min;
                4. Mounting media, read slides. Count stained with
DAPI-mounting media.
                 5. For double staining, I did the same thing, except
using mixed first antibodies and mixed second conjugated antibodies.

Designing: Stain 1: Rabbit anti-mouse antibody, and donkey anti-rabbit
conjugated with Alexa Fluor 488;
                Stain 2: Rat anti-mouse antibody, and donkey anti-rat
conjugated Alexa fluor 594;   

My problems:  The individual staining signals are good. But there is
some match color between two channels. For example, I could see weak and
similar green signal when I applied Alexa 594. Basically, I could see
the weak but true alveoli structure of mouse                            
     lung.
                       Then I performed double staining, I could see
lots of signal match between red and green channels, which should not be
true. In this way, I could see basic alveoli structure of mouse         
                        lung in both green and red channels. By the way,
I tried to red the slides before staining and after blocking, the
autofluorescence is very very weak.

I am really frustrating with this situations, and please feel free to
tell me how I could figure out this problem. Any of your suggestions and
comments are very much appreciated.

Thanks,



~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
Chengming Wang 
DVM, M.S., Ph.D. 
Department of Pathobiology
College of Veterinary Medicine
Auburn University
264 Greene Hall
Auburn, AL 36849-5519

Voice: (334) 844-2624
Email:  wangche <@t> auburn.edu