Re: [Histonet] RE: Antibody staining on non-pfa fixed tissue

From:Ruth Yaskovich

I have had good results with mouse and rat brain fixed in 70% Alcohol.
Ruth Yaskovich
National Institutes of Health
National Institute of Dental and Craniofacial Research
Pain and Neurosensory Mechanism Branch

On 7/31/06 3:56 AM, "C.M. van der Loos"  wrote:

>    Dear Greg,
>    As you already concluded the epitope of interest is PFA-sensitive. How
>    about  using  a  non-crosslinking fixative here? For example methacarn
>    (methanol:chloroform:acetic    aced    =   6:3:1) does   not   contain
>    formaldehydes.  Fix  your  sample  for 48 hours, dehydrate via alcohol
>    series  and  embed  in paraffin. Because methacarn is non-crosslinking
>    fixative most antigens doesn't need antigen retrieval.
>    Just a suggestion...
>    Chris van der Loos, PhD
>    Dept. of Pathology
>    Academic Medical Center M2-230
>    Meibergdreef 9
>    NL-1105 AZ Amsterdam
>    The Netherlands
>    Date: Fri, 28 Jul 2006 18:46:26 +0200
>    From: "Gregory A. O'Sullivan" 
>    Subject: [Histonet] Antibody staining on non-pfa fixed tissue
>    To: "HistoNet" 
>    Hello HistoNetters,
>    I  realise  that  this  may  be a question that has arisen on previous
>    occasions, however, I have been unable
>    to find an exact answer from a search of previous email correspondence
>    on HistoNet.
>    I  would  like to detect and examine the distribution of my protein of
>    interest in mouse brain sections
>    (with  particular emphasis on the hippocampus) and compare it to other
>    known markers.
>    The  problem is that the antibody I am using is extremely sensitive to
>    pfa fixation but the marker antigens
>    are  best  detected  on  fixed  tissue.  I have tried to overcome this
>    problem by fixing cryostat sections
>    briefly  with  pfa  before using different antigen retrieval protocols
>    (either microwave or heated bath incubatio! n
>    in C
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