[Histonet] Re: Pink precipitate... from BCIP/NBT reaction on ISH?

[Gayle Callis]

We have used DAKO's ready to use BCIP/NBT and always develop the color 
using a microscope.  This particular product is fast, with color 
development in a shorter time, but that is the differences we experience as 
compared to Teri's longer time in the substrate.   So while you are 
developing the color, be sure to monitor its progress with a microscope, 
pull your positive and look at it at different time increments, you can 
return it to the chromogen to proceed to color endpoint if not dark 
enough.  We develop with this DAKO product on slides laying flat, but are 
careful to have microscopic monitoring, and do not experience ppt with this 
ready to use.

Teri's suggestion is excellent and one that we use with AEC, the van der 
Loos way, so as to not get ppt on sections with that chromogen 
either.  Vertical slides in a coplin jar and or slide mailer works too, but 
we do pull the positive for monitoring color development.

Not only less time in chromogen but also check the concentration of your 
primary, if your color is coming up way too fast with too dark a ppt, you 
may need to adjust your primary concentration - BCIP/NBT is very sensitive, 
up there with DAB.  Hopefully you did a dilution panel of the primary for 
optimal working concentration with this chromogen.

At 07:37 AM 9/27/2005, you wrote:
>Ralph,
>
>When we use BCIP/NBT, we do our color reaction development in the 37
>degree C incubator in the dark.  We change the solution frequently
>(every several hours, or when the solution turns pink).  We do not lay
>the slides flat for their incubation, instead we use the 5-slide plastic
>mailers and have them standing upright.  This keeps precipitate from
>settling on to the glass.  Many of the chromogens for Alk. Phos. tend to
>form a precipitate, and the manufacturers don't recommend filtering.
>
>Your signal appears to be quite strong.  Perhaps you can also decrease
>the time in chromogen?
>
>Best of luck to you,
>
>Teri Johnson
>Managing Director Histology Facility
>Stowers Institute for Medical Research
>1000 E. 50th St.
>Kansas City, MO 64133
>
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Gayle Callis
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610
406 994-6367
406 994-4303 (FAX)