Can someone here share their experience with 5HT2A and SERT immunostaining on rat brains. I have tried to stain by several different procedures, but unfortunately I was unsuccessful.
Can you explain what are the different procedures you employed?
Hi,
Rat brain tissue was isolated after perfusion with PBS followed by 4% PFA. After two days of isolation, 4% PFA was replaced with 2% PFA and two days after dehydrated for two days with 30% sucrose solution. Slices of 16 micrometers were made.
In the first instance slices were prefixated with 4% PFA and other batch without prefixation.
After prefixation or no prefixation two batches were run, one with antigen retrieval and another without antigen retrieval (sodium citrat buffer with 0.01% tween 20, heated for 10 mins).
Slides were blocked using 10% NGS for 1 hour and then incubated with primary antibodies for overnight at 6 degree Celsius(5HT2A: 1:100, 1:200, 1:300 Ab16028; SERT:1:2500, 1:5000, 1:7500, Ab79328).
on day 2 slides washed and incubated with secondary antibody for 2 hours at a dilution of 1:250.
In the antigen retrieved batch I see all background for 5HT2A, and very little staining without antigen retrieval, however for SERT no staining in any case.
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