[Histonet] CD-3 woes

[Favara, Cynthia (NIH/NIAID) [E]]

I have been using Dako CD-3 on murine formalin fixed paraffin embedded
tissue and am experiencing some variation in staining. My protocol:
pretreatment with Citrate pH 6.0 @120C for 5 minutes under pressure, an
overnight incubation of the primary 1:1500 @4C, followed by 30 minute
incubation with Vector biotinylated anti Rabbit IgG 1:250, 30 minutes
Biogenex SS Streptavidin all steps following primary are done on the
Ventana Nexus @RT using their AEC. I used to do the stain reliably but
have a new lot and reliability is not optimal. I use a mouse spleen and
brain with T-cell infiltration as positive controls and a NL brain as a
negative control. Sometimes the stain is so crisp and other times it is
muddy with a decrease in the number of cells stained. I have checked lot
numbers on all regents and they are the same. It has occurred to me that
doing the peroxidase block following the primary could be problematic
however I do not see the same problem with other antibodies treated the
same way and seem to recall that peroxidase block is done by many
following the primary incubation. I have tried different buffers for
pretreatment and also PK. Any suggestions/ commiserations  would be
welcome.

 

Thanks,

c

 

Cynthia Favara 
NIAID/NIH/RML/LPVD 
903 South 4th Street 
Hamilton, MT 59840 
406-363-9317 

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