[Histonet] NTMT woes

[John Kiernan]

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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