Immunostaining for frozen sections

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Guy Sovak
Guy Sovak's picture
Immunostaining for frozen sections

Immunostaining with fluorescent secondary antibodies for frozen sections
1. Prepare frozen sections from your tissue
2. Mount it on L-PolyLysine Slides.
3. Wash sections for 30 seconds in cold Acetone (-20°C). This step allows better antibody penetration (may wash out your antigen, especially in the case of cytosolic proteins). Let section dry before next step (in hood).
3. block non-specific binding by incubation of section with 1%-0.5% goat serum in PBS for 1 hour at 37°C.
4. Draw circle round dry section with PAP pen to prevent loss of antibody. Add primary antibodies; Typically dilute primaries to 1mg/ml and make up in PBS plus 0.1% goat serum. Usualy for double staining you can apply chicken, rabbit and mouse antibodies at same time. I prefer to do it setep by step.Typically incubate for 1 hour at 37°C or 2 hours at room temperature or overnight at 4°C.
5. Wash sections at least 3 times, at least 5 minutes each time, in PBS. To reduce background can include 0.1% Tween 20 in PBS.
6. Apply secondary antibodies. Best are goat anti-mouse and goat anti-rabbit antibodies from Molecular Probes, or Jackson. Typically incubate for 1hr at 37°C or 2 hours at room temperature at a dilution 1:5000.
7. Wash sections at least 3 times, at least 5 minutes each time, in PBS.
8. Mount in mounting medium, a useful one being Vectasheild mounting medium with DAPI, which allows you to stain nuclei with the DNA intercalating fluorescent dye DAPI, which you can see on a fluorescence microscope fitted with appropriate blue filters.
9. View on fluorescence microscope.

vilsy
vilsy's picture
Hi,

Hi,
I just cannot udnerstand why wou would want to do the double stainign sequentially rather than together. It seems like by staining wiht two antibodies at the same time.,, difference in species will lead to more competition and hence less non-specific binding...am I wrong?

Guy Sovak
Guy Sovak's picture
Normal

Normal
0

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That may be true
But I had some experience in which I did double staining with different ab' and did not get good results. When I did it sequentially the results were much better. Why, I do not have a good answer.
Thanks
 
Guy

Arvind Singh Pundir
Arvind Singh Pundir's picture
please go through the

please go through the following article :  Multiple staining in Immunohistochemistry
which describes about Antibody cocktails,Double staining,sequential double staining,Simultaneous double staining
 References:  Van der Loos, C. M. Immunoenzyme Multiple Staining Methods. New York: Springer-Verlag, 1999. 
www.mihisto.org/pdf/tp/tp.2006.06.pdf