When you do double staining you should try the following.
1. Change the order of primaries try first BrdU and second doublecortin.
2. You can also try adding two primaries together (simultaneous
staining) and also two secondaries together.
3. After each staining quickly check the slides under the microscope
for any signal without mounting them. If you have signal proceed with
the second staining.
4. After you do the pretreatment for the second staining also quickly
check the slides If the signal from the first staining is still there.
In most cases retrieval of one staining interferes with the second
epitope. If pretreatment of one antibody masks the other try to
optimize one of the antibodies with a pretreatment that does not
affect the other or use antibodies from different
5. For double staining try to use secondary antibodies that are design
for multiple staining. They are re adsorbed for cross reactivity with
other species. Sometimes they work.
6. Finally NEVER dehydrate and clear immunofluorescence slides with
alcohols and xylene. You should mount the IF slides with glycerol
based anti-fade media and keep the slides at -20 C horizontally in a
slide folder. I use 0.1 % para-phenylenediamine in 80% glycerol in PBS
for mounting. There are also different types of commercially available
anti-fade mounting media for IF slides.
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