Immunohistochemistry - no positive staining - (Jan/17/2005 )

hi all,
i have been doing immunohistochemistry for eight months.i am using polyclonal antibody as primary,we isolated it from rabbits.at first i got nice results with non-positive tissues. the problem was false positive staining,which i reduced thru blocking with normal, goat sera,as we get DAKO secondary antibody from goat.

now for the last four months i am not even getting a faint color on strictly positive tissues.i have tried to reduce the blocking,no results, used fresh antibody ,secondary antibody incubation time also increased,also without any results.please help .

Is it the same batch of polyclonal Ab? Is this serum or is it affinity purified in some way? Has it been stored properly? Are you sure it is still active? Are your sections fresh? Were they stored properly? Are your secondaries still working? Were they stored properly? You can run a battery of tests to prove this but my feeling is the pAb is losing it's activity or degrading etc over time.

If you provide more details of your protocol I can probably try and help.