Re: NADPHd artifact
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From: | Mary Latimer <ml4@st-andrews.ac.uk> |
To: | Susan Travers <travers.3@osu.edu> |
Reply-To: | |
Content-Type: | TEXT/PLAIN; charset=US-ASCII |
Dear Susan I have done a lot of NADPH stains and i think that it is not
stable in Xylene so i use glycerol gelatine as mounting media...my
experience is that they appear to break down after a few weeks but stuff
mounted in glcerol gel is good 5 years on...hope this helps Mary
On Fri, 18 Feb 2000, Susan Travers wrote:
> I am writing to ask if anyone has seen and has a solution to an artifact
> that we have seen in our NADPHd material. It appears as a small, elongated
> crystalline precipitate that appears to be on the surface of the tissue.
> Our technique for doing the NADPHd is the standard one using .1% bNADPH &
> .01% nitroblue tetrazolium in PB. The only twists are: that the tissue is
> fixed with PLP , and we double-label the tissue using DAB
> immunohistochemistry (first NADPHd, then ICC)
>
> We have tried to trouble shoot by (1) filtering the staining solution-- no
> help, (2) doing the reaction in the dark (inconvenient and no help), (3)
> exploring the effects of using ETOH to dehydrate or not-- this is
> interesting-- with ETOH, which is our standard procedure the artifact looks
> crystalline, without ETOH-- looks like very small spherical globules, (4)
> using xylene or HEMO-D to clear-- no effect, and (5)coverslipping with
> permount versus cytoseal (no effect).
>
> The only other hints are that we never see the artifact on wet,
> uncoverslipped sections, and occasionally it seems that the artifact gets
> worse the first few days after coverslipping; also the artifact does seem
> worse in the double-labeled tissue.
>
> Any advice would be greatly appreciated.
>
>
>
> 'Regards,
> Susan Travers
>
>
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