[Histonet] Extracellular matrix proteins fixation. Details

[Diego J. Rodríguez Gil]

Hi:

     Thanks a lot to all of you who answer!. Since some of you asked, I am
sending some more details.
     I am working on mice (CD-1), and the molecules I am trying to stain are
named Wnt (Wingless-Int). Antibodies are from R&D systems (most of them are
goats), and there are no previous works reporting the use of these antibodies.
I am only making the assumption that they work, based on the datasheet the
company provides.  I could see some very faint positive stain for some of them,
but I am sure I losing some because of fixation. Besides that, I know they are
expressed based on RT-PCR and in situ hybridization experiments.
     Brief protocol is:
         Perfusion with 4% PFA, and then overnight deep in 4% PFA at 4 C.
Transfer to PBS, then 30% sucrose til they sink, freeze on OCT and then section
on cryostat at -18C.
         The antigen retrieval protocols that I tried were steaming (2, 5 and 10
min with sodium citrate) or pepsin in HCl. None of them gave me any better
stainings.
         Staining protocol: Wash in TBS and then add 2% BSA in TBS-T, for 30
min. Primary antibody (on 2% BSA) Over night .
	Wash 3 x TBS-T.
	Add secondary antibody on 2% BSA for an hour.
	Wash 2 x TBS-T.
	Wash TBS and mount with Crystal Mount.

      Thanks again!
      Best regards,
      Diego.

-- 
Diego J. Rodríguez Gil
Postdoctoral Associate
Department of Neurosurgery
Yale University School of Medicine
PO Box 208082
333 Cedar St, FMB-430
New Haven CT, 06520-8082
U.S.A.