[Histonet] Fixatives for IHF

[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 
EtOH).

Any input will be welcomed.

Alejandro


-- 
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