[Histonet] Fixatives for IHF

From:Alejandro Ortiz Stern

Hi all,

I am working on mouse lung, and have come across a small problem.

Basically I embed the lungs in OCT/sucrose with snap freezing in 
Isopentane and then postfix.

Initially I was postfixing in pure acetone but there staining with 
certain antibodies was not working

We are trying to detect: DAPI, Smooth muscle actin (directly labeled 
PE), CD4 (Directly labeled Alexa 647), Thy1.1 (FITC coupled + GARabitt 
Alexa 488) and Thy1.2 (Biotinylated + Stv Alexa 594)

Now the conflict:

When I postfix with 100% EtOH I get a very nice staining with Thy1.1 and 
CD4 but have not managed to see the Thy1.1 and the SMA looks terrible

When I postfix with either Acetone or PAF 1% the SMA look lovely but the 
CD4 staining is very dull.

I have not managed yet to detect the Stv Alexa 594 signal yet.

How can I get the best of both worlds? A very nice and well demarcated 
SMA staining (as with PAF of Acetone) with a nice CD4 staining (as with 

Any input will be welcomed.


Alejandro Ortiz Stern M.D. Ph.D.,
Postdoctoral Fellow
Institut National de la Santé et de la Recherche Médicale, E0344
Université de Nice-Sophia-Antipolis
Institut de Pharmacologie Moléculaire et Cellulaire
660 Route des Lucioles - 06560 Valbonne - FRANCE
TEL: 33 (4) 93 95 77 80 - FAX: 33 (4) 93 95 77 08

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