Only 1/50 ml (20 l) of Water-soluble Aminoplastic Resin Is Enough to Embed One Biological Sample for EM Study of Membrane and

Lipid Ultrastructure
Johnson Gao
Retired professor in Cell Biology 8/8/2013 e-mail: jkxgao@gmail.com

It is a shame that the lipid ultrastructural research is far less developed than the ultra-structural researches of protein, polysaccharide, and nucleic acid based subcellular compartments, although all of those four substances are the common structural building materials and they almost share equal importance to the existence of living things. The delay in lipid ultra-structural research is mainly due to the lack of a proper water-soluble embedding medium commercially available on the market that can avoid organic solvents for dehydrating and infiltration agent, which will extract fat and lipid. If someone tells you that only 1/50 ml (20 l) of water-soluble resin is enough to run the infiltration and embedding of biological sample in one single step for electron microscopic study of ultrastructure of lipid, you may say: Are you teasing? Is it really possible that so little amount of embedding medium can do the job? Or, you may say: It is fantastic. It is super saving. Tell me how to do with that kind of embedding, immediately. Tell you the truth. The technique had

existed for long time, which was in the year 1982. It was called as silylated aminoplastic embedding method.[1] It had nicely shown good result in the study of lipovitalline molecule changes in the first cleavage of frog. And, with that method, I had published the worlds first batch of color electron [2] micrograph. The aim of my work at that time was that I wanted to join a fundamental discussion - If the human being was created by the God, or, through the long course of evolution? and I heard that Friedrich Engels had a famous saying about life and chemistry (1894). His definition reads: Life is the existence form of proteic structures, and this existence form consists essentially in the constant self-renewal of the chemical components of these structures. Unfortunately, the publication of my research was written in Chinese. So, my method was not known by most of American scientists. Recently, to reduce the difficulty caused by language barrier, I had described that method in English in a mini review. [3] In that small pamphlet, four main steps of silylated aminoplastic embedding method are dually presented.

Since that aminoplastic resin is not commercialized yet, if you want to try it and to verify whether 20 l of water-soluble aminoplastic is really enough to embed you sample for EM study of lipid with your own project, you have to synthesis the resin by yourself. Here is the operation instruction for you. First, dissolve 30 g of paraformaldehyde and 10 g of urea in 30 ml of 25 % glutaraldehyde in a beaker and 7 ml of distilled water is added. Adjust the pH to 8.0 by adding few drops of 25~28% ammonium water. Transfer the mixture into a reflux and keep it at 80 C for 3-5 hour until the mixture turns clear. Because the aminoplastic will cause selfpolymerization at low pH condition, care must be taken that the reaction in the first hour may cause a drop of pH to 6. If you do not use the ammonium water (about 50 drops) to bring it back to the weak alkaline condition and adjust the pH to 7.5, the whole mixture may cause polymerization and become useless. Second, prepare the working medium for infiltration and embedding. It is composed of 400 l of aminoplastic solution, 60 l of PEG-400 and 20 l of 30% NH4Cl. Adjust the mixture to pH 4

with HCl. And now 20 l of that working medium is enough to do the infiltration and dehydration for one sample, which is performed in a combined step in a plastic ring about 2 mm inner diameter and 2 mm in height at 4 C overnight in a mini desiccator, using P2O5 as an effective dehydrating agent. Of course, you must trim your sample to the size of 1/4 mm3, or, 1/8 mm3. Polymerization of the resin is performed at 37 C for 30 min. Then, cut out the unrelated part of cured resin. The sample shall be followed by 60 C incubation, for overnight, to increase its cross-linking.[1, 3] You must hook a silylation technique to transform all of -OH groups in the solidified resin block to the hydrophobic organic silicon residues[4] that repels water effectively and that makes cutting for easy. The tools needed for silylating procedure are shown in the diagram 2 in the literature cited No. 3. Finally, the sample must be mounted on a plastic rod for sectioning.[3] The thin sections can be stained with normal EM section staining. Anyone who is interested in develop a kit of silylated aminoplastic embedding may touch me for further know-how knowledge.

Literature cited
[1]. Gao, K. X. and M. Y. Zhou (1982) Silylated aminoplastic used as a water soluble embedding medium for electron microscopic study of lipids. Acta Biologiae Experimentalis Sinica., 15(1): 57-65. [2]. Gao, K. X. and K.Y. Ku (1983) Ultrastructural changes of yolk platelets during the first cleavage of the fertilized eggs of Rana amurensis. Acta Experimentalis Sinica, 16(3): 325-337. [3]. Gao, Johnson (2013) A Mini Review of Water-soluble Aminoplastic for Electron Microscopy: The Story Behind the World's First Color Electro Micrograph. http://www.lulu.com/shop/johnsongao/a-mini-review-of-water-solubleaminoplastic-for-electron-microscopythe-story-behind-the-worlds-first-colorelectro-micrograph/paperback/product21127316.html [4]. Chambaz, E. M. and E. C. Horning (1969) Conversion of steroids to trimethyl derivatives for gas phase analytical studies: Reaction of silylating reagents. Anal. Biochem., 30: 7-24.