Counterstain for AEC AND BCIP/NBT

Dr Engelbert Buxbaum via methods%40net.bio.net (by engelbert_buxbaum from hotmail.com)
Fri Sep 5 14:11:33 EST 2008


Am 28.08.2008, 03:06 Uhr, schrieb Bluescript <shauke from uni-bremen.de>:

> On 27 Aug., 21:10, "Dr Engelbert Buxbaum"
> <engelbert_buxb... from hotmail.com> wrote:
>> Am 27.08.2008, 04:30 Uhr, schrieb Bluescript <sha... from uni-bremen.de>:
>>
>> > Hi,
>> > I want to do immunohistochemistry detecting two targets at the same
>> > time. For various reasons I am going to use AEC as substrate for HRP
>> > and BCIP/NBT as AP substrate. Now I am looking for a good
>> > counterstain. Because of the AEC the counterstain has to work with an
>> > aqueous embedding medium.

> Thank you very much for your suggestions. Maybe I forgot to mention
> that I am looking for a chromogenic counterstain that can be
> visualized by light microscopy!

Classical nuclear stains like haematoxilin, methylgreen, nuclear fast red  
should work, perhaps you'll have to apply them before immunostaining.  
Methylenblue-eosinate (Giemsa, Wright, Papenheim...) would give blue  
nuclei and pink cytosol, you'll have to check whether the contrast with  
the immunostaining is sufficient. Perhaps switch to  
Diaminobenzidine/Nickel for HRP detection (black, chemically quite inert).  
But overall, I'd recommend swithching to fluorescence detection, check
http://probes.invitrogen.com/handbook/


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