[Histonet] Michel's transport medium

=?gb2312?B?1qO0us+8?= springrosycloud <@t> 126.com
Wed Jan 31 06:40:01 CST 2007

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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