[Histonet] My frustrating experience with double fluorescence staining!

From:"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                            
                       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.


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@auburn.edu

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