RE:In situ with AP and Fast Red TR/Naphthol
Did you consider the staining sensitivity/efficiency of the mentioned
chromogens? For example, Fast Red TR is far less sensitive/efficient than
NBT/BCIP. In other words are you sure that your signal will be visible
after development with Fast Red?
Indeed, the reaction product of Fast Red TR/naphtohol-AS-MX-phosphate will
dissolve in alcohol and/or xylene. However, there is a new Dako Fast Red
kit available that is more sensitive/efficient than the regular Fast Red
TR/naphthol-AS-MX-P visualization and that can be mounted up organically. I
don't have the order number over here (it's not mentioned in the 2002
catalog). Furthermore, there is Vector Red, an AP substrate/chromogen that
can also be mounted up organically
Alternatively, you may use the fluorescent properties of Fast Red and
Vector Red (New Fuchsin is not working for this). When applying the
TRITC-filter pack in your fluorescence microscope, just minute amounts of
Fast Red reaction product will give a very strong bright red fluorescence
image. Fading of this signal is about minimal when compared with FITC or
TRITC fluorescence. This fluorescent approach may circumvents the problem
with the blackish material.
In response to the idea to use New Fuchsin instead, I believe that Fast Red
TR will result into a more crisp staining than New Fuchsin.
Chris van der Loos
Academic Medical Center
Dept. of Cardiovascular Pathology
NL-1105 AZ Amsterdam
>Date: 19 Feb 2003 09:15:49 -0600
>From: Lisa C Ranford Cartwright
>Subject: In situ with AP and Fast Red TR/Naphthol
>Hello histo folk
>We are performing in situ PCR on tissue sections (fixed, embedded,
>sectioned, etc). The PCR results in DIG incorporation into the DNA, which
>we then detect with an anti-DIG antibody conjugated to alkaline
>phosphatase. This part works fine (using a fluorescent detection system).
>We now wish to use a colorimetric substrate rather than a fluorescent one,
>and FAST Red TR/Naphthol had been suggested. This is supposed to give a
>bright red stain. However we also wish to counterstain with Giemsa, and I
>understand the Fast Red is soluble in organics. The final methanol
>concentration in the Giemsa stain will of course be low (5% stain) - is
>this likely to dissolve the fast red deposits?
>I've never used FAst Red TR before. Has anyone tried Fast Red TR and Giemsa
>together? Any thoughts on whether it will work? Any alternatives (note we
>don't want to use NBT/BCIP substrate as our tissue already as some blackish
>material in it which we think will be confused with the signal).
>Thanks for any suggestions, comments and assistance.
>Division of Infection & Immunity
>Institute of Biomedical & Life Sciences
>Joseph Black Building
>University of Glasgow
>Joseph Black Building, Glasgow G12 8QQ
>Tel: 00 44 141 330 2639
>Fax: 00 44 141 330 4600
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