Re: [Histonet] removing excess x-gal stain (LONG)

From:John Kiernan

Dear Linda K Bauer,

Nobody has replied to you on Histonet for about 
a week, so a few speculative comments from a 
non-user of the X-gal technique may be better 
than nothing at all. This reply has three parts.

1. How can you have "excess nonspecific X-gal
stain"? The substrate (X-gal) is hydrolysed by
beta-galactosidase and one of the products of
hydolysis, "X" (which is short for
5-bromo-4-chloroindoxyl), is rapidly oxidized (by
the ferricyanide ions in the incubation medium) to
an indigoid dye that rejoices in the name of
5,5'-dibromo-4,4'-dichloroindigo. This particular
indigoid dye sticks strongly to the nearest
protein molecules and does not diffuse into fat or
other lipids, and does not form visible crystals.
   This is one of the most thoroughly studied 
reactions in enzyme histochemistry. If the method
was carried out correctly there should be no
"nonspecific" deposition of 
5,5'-dibromo-4,4'-dichloroindigo more than 500nm
from a beta-galactosidase enzyme molecule.

   There are some minor controversies about the
sensitivity and specificity of indigogenic methods
for lysosomal enzymes (with acidic pH optima), and
for many of these enzymes there are alternative
histochemical methods. 

2. I'm assuming that you are using the indigogenic
X-gal method to detect the lac-Z reporter gene,
which encodes a bacterial beta-galactosidase that
works at pH 7+. This gene is tagged onto genes
that encode more interesting proteins in
experiments with altered cell lines, transgenic
mice etc. Cells that transcribe and translate an
added or modified gene also make the bacterial
enzyme encoded by the reporter gene. Histochemical
detection of a beta-galactosidase active at ph 7+
indicates that the "more interesting proteins" are
probably being made in the same cell.
   The problem with lysosomal enzymes referred to
at the end of Part 1 (above) does not arise with
indigogenic detection of the lac-Z gene product
because the pH of the medium is significantly
higher than the pH optima of lysosomal
"Normal" (animal) beta-galactosidase activity
would be detected only after an unduly prolonged
incubation. The false-positive result would also 
show up in your negative control sections of 
tissues known not to express the lac-Z gene.
   If your known-negative controls show no 
blue-green colour and your transgenics (or others)
show "excess nonspecific X-gal stain" there is no 
"excess". You must be working with a tissue whose 
cells are putting out lots of the bacterial 
enzyme. The genetic engineer should thank you for 
providing evidence that the introduced genes 
entered the host organism and were successfully 
transcribed and translated.
   If you have no known-negative and 
known-positive control results, ask your boss 
for more detailed advice. 

3. This is an answer to your beautifully concise 
two-line question, cited below. The indigoid dye
is insoluble. It was probably deposited at the 
site of the enzyme, if the histochemical method 
was done by the book. 

John Kiernan
Anatomy, UWO
London, Canada
Linda K Bauer wrote:
> I would like to remove excess nonspecific x-gal stain from whole mount
> murine embryos. Does anyone have a suggestion or technique for this?
> Linda(Lin)Bauer
> Department of Genetics, Cell Biology, and Anatomy
> 985455 Nebraska Medical Center
> Omaha, NE 68198-5455
> Phone: (402) 559-2863
> Fax:   (402) 559-4001
> Email:
> _______________________________________________
> Histonet mailing list

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