Re: [Histonet] Michel's transport medium

From:Rene J Buesa

I used to prepare Michel's solution in the following way:
  sol. 1- 1 M sol. of potassium citrate monohydrate----- 32.42 g/100mL dist. water
  sol. 2- 0.1 M sol. of magnesium sulfate hepta hydrated -------2.46 g/100 mL
  sol. 3- 0.1 M sol. of N-ethyl maleimide ---1.25 g/100 mL
   
  To prepare 1 litre of Michel's add:
  a- 25 mL of solution 1
  b- 50 mL of solution 2
  c- 50 mL of solution 3
  d- 875 mL of distilled water, and
  e- 550 g of ammonium sulfate
  f- when all components are dissolved adjust pH to 7.0 using a 1M solution of potassium hydroxide.
  The solution should be kept between 0 and 6C
  This procedure always worked fine with me. Try to use analysis grade chemicals.
  Hope this will help you.
  Ren J.

֣ϼ  wrote:
  
Hello, everyone. I am going to use Michel's transport medium to send renal tissue samples. I got a protocol on the medium preparations(see belows). When I made the preparation according to the protocol, I met troubles. At the first step(1.0 M potassium citrate buffer pH 7.0), I could not adjust pH using the 1M potassium hydroxide. I had to adjust pH to 7.0 using 10M potassium hydroxide(40ml). And then, I could not dissolve 55 grams of ammonium sulfate in 100 ml washing solution. About half of ammonium sulfate(made in China) kept unsolved, even though I incubated it in heat water and/or mechanical stirring. When I bought a bottle of new ammonium sulfate, it still did not work. Later, I tried to dissolve 55g ammonium sulfate in 100ml distilled water, but I failed. I tried and thought it again and again. I did not know what is the matter. Please tell me what I should do. I am waiting for your kindly reply. Sincerely, Betty 
Nanjing University School of Medicine, 
Research institute of Nephrology, Jinling Hospital, 
Nanjing, China 210002
Tel: 86-025-81765894
Fax: 86-025-84801992The protocol from http://publish.uwo.ca/~jkiernan/faqlist.htm#MICHELammonium sulfate was unsolved. 1.0 M potassium citrate buffer pH 7.0: dissolve 21.0 g citric acid monohydrate (or 19.2 g citric acid anhydrous) in 30 mL of hot deionized or distilled water. Cool. Adjust pH to 7.0 with 1 M potassium hydroxide (about 35 mL) Dilute to 100 mL with more water. Washing solution: 25 mL 1.0 M potassium citrate buffer 50 mL 0.1 M magnesium sulfate heptahydrate (F.W. 246.5) 50 mL 0.1 M N-ethyl maleimide (= 12.5 g in 1 L of water) (Sigma E3876.) Water to make 1 L Adjust to pH 7.0 with 1 M potassium hydroxide Store in refrigerator. (Cost about $50/25 g in 1994.) Transport medium: Dissolve 55 grams of ammonium sulfate in 100 mL washing solution. (Add slowly, with mechanical stirring.) Adjust pH to about 6.9 with 1 M potassium hydroxide (< 2 mL needed)Reference: Michel B. Milner Y. David K. Preservation of tissue-fixed immunoglobulins in skin biopsies of patients with
 lupus erythematosus and bullous diseases. A preliminary report. J. Invest. Dermatol. 59: 449-452 (1973).



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