[Histonet] Extracellular matrix proteins fixation. Details

From:"Diego J. =?iso-8859-1?b?Um9kcu1ndWV6?= Gil"


     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
         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 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

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