[Histonet] NTMT woes

[Johnson, Teri]

Good point, John. I will have my pH verified by our media prep lab.
 
NTMT are the first initials in the materials used to make it (NaCl +
Tris-HCl + Magnesium chloride + Tween). 
 
We are using it as not only buffer rinses prior to chromogen substrate
incubation, but also as the diluent for the NBT (Nitro blue tetrazolium
chloride; C40H30Cl2N10O6; MW 817.7) and BCIP (5-Bromo-4-chloro-3-indolyl
phosphate, toluidine salt; C8H6NO4BrCIP X C7H9N; MW 433.6).
 
I will also be reordering the Magnesium chloride in a smaller amount -
I'm wondering if ours is too saturated with water already (one month
old).
 
Teri

	-----Original Message-----
	From: John Kiernan [mailto:jkiernan <@t> uwo.ca] 
	Sent: Thursday, September 20, 2007 10:21 AM
	To: histonet <@t> lists.utsouthwestern.edu; Johnson, Teri
	Subject: Re: [Histonet] NTMT woes
	
	
	The precipitate must surely be magnesium hydroxide, as a
consequence of the pH being too high. Precipitation is to be expected in
an alkaline solution containing magnesium ions  Data in the Merck Index
indicate that Mg(OH)2 becomes insoluble at pH 9.5, which is close to the
pH 9.3 of your solution. A small error in pH measurement, due to
deterioration of the meter's electrode or of one of the buffers used for
calibration, might result in a solution that is too alkaline. 
	 
	Curiousity. What does NTMT stand for? Google reveals this
solution as an unexplained rinse to follow in situ hybridization, prior
to what might be a histochemical reaction for an alkaline phosphatase
label. Strongly alkaline rinses do not figure in the techniques given in
the RMS In Situ Hybridization Handbook (Leitch et al, 1994). 
	
	John Kiernan
	Anatomy, UWO
	London, Canada
	---
	----- Original Message -----
	From: "Johnson, Teri" <TJJ <@t> Stowers-Institute.org>
	Date: Thursday, September 20, 2007 10:33
	Subject: [Histonet] NTMT woes
	To: histonet <@t> lists.utsouthwestern.edu
	
	> We're really stumped on what's going on here. For years we
have made
	> this solution without any trouble or issue. Suddenly in the
past 
	> severalmonths, we're having trouble with it going cloudy one
day 
	> after making
	> it. I made some yesterday, and by this morning it was cloudy.
I 
	> checkedthe pH and it was 9.3 so I'm not too worried that it's
a 
	> pH issue. This
	> cloudiness occurs regardless of whether we use Tris base and
pH with
	> HCl, or if we use Tris-HCl and pH with sodium hydroxide.
	> 
	> Here is the recipe:
	> 
	> NTMT (500 ml)
	> NaCl 5M............................................10 ml
	> TrisHCl, 2M, pH 9.5................................25 ml
	> MgCl2 2M.........................................12.5 ml
	> Tween-20 (10% in dd water).........................50 ml
	> q.s. to 500 ml (store at RT)
	> 
	> 
	> NaCl 5M (100 ml)
	> NaCl (FW 58.44)....................29.22 g
	> q.s. to 100 ml (store at RT)
	> 
	> TrisHCl, 2M (500 ml)
	> Tris Base (FW 121.1).............121.1 g
	> Dissolve Tris base in 300 ml of d water. pH to 9.5 with HCl.
	> q.s. to 500 ml (store at RT)
	> 
	> MgCl2, 2M (100 ml)
	> MgCl2 (FW 203.3)..................40.6 g
	> q.s. to 100 ml (store at RT)
	> 
	> If we omit the magnesium chloride, it stays clear. If we add
it, 
	> by the
	> next day it is very cloudy and cannot use the solution.
	> 
	> Can anybody give me any information on what might be
happening, 
	> and what
	> we need to do? We have already replaced the magnesium
chloride, 
	> thinkingit had gone bad somehow. It's still happening.
	> 
	> Thanks for any help you can give us.
	> 
	> Teri Johnson, HT(ASCP)QIHC
	> Managing Director Histology Facility
	> Stowers Institute for Medical Research
	> 1000 E. 50th St.
	> Kansas City, MO 64110
	> 
	> 
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