Chemicals for Electron and Light Microscopy

Acetonitrile

Suggested substitute for Propylene Oxide because it is reported as noncarcinogenic, less toxic and not as flammable. High purity; miscible with water, alcohols, acetone and epoxy resins15.

15) Tarnowski BI, Schonbaum GR: Acetonitrile: substitute for propylene oxide in tissue processing for transmission electron microscopy. Proc 42nd Ann Meeting Elec Mic Soc Amer, p 38, 1984

Edwards HH, Yeh YY, Tarnowski BI, Schonbaum GR: Acetonitrile as a substitute for ethanol/propylene oxide in tissue processing for transmission electron microscopy: Comparison of fine structure and lipid solubility in mouse liver, kidney, and intestine. Micro Res & Tech, 21, pp 39­50, 1992

Also used as a dehydrating agent for cells prepared for SEM: Holshek JG, Akins RE: Acetonitrile is better than ethanol as a dehydrating agent for cells prepared for SEM. Proc Mic Soc of Amer, 52nd Annual Mtg 1994, San Francisco Press, ed GW Bailey and AJ Garrett-Reed, pp 324-325, 1994

(Rosaniline Hypochloride; Magenta; Basic Violet 14)
Berkowitz et al., (1968). Selective staining of nervous tissue for light microscopy following preparation for electron microscopy. J. Histochem. Cytochem. 16:808. Sato, T., and Shamoto, M., (1973). A simple rapid polychrome stain for epoxy embedded tissue. Stain Technol. 48:222.

Contains anti-fading solution. Suitable for preventing fading of dyes such as DAPI which have the tendency to disperse in glycerol based mounting solutions.

Colloidal Adhesives: See "Adhesives"

Histological Fixative. Phosphate Buffer. pH 6.9 - 7.1 at 25°C.

Note: ACS Reagent Grade Formalin (35 - 38% Formaldehyde, 10 - 15% Methanol) larger quantities available on request.

EM grade purified glass distilled Glutaraldehyde sealed with inert gas in amber small-volume ampules, or in bottles for larger volumes.

Resin with catalyst packaged separately. Accelerator 18185 can be used for cold-cure (room temperature). Heat cure is 24 hours between 50°C and 65°C. Gelatin capsules are recommended for temperatures below 60°C. Tightly closed BEEM® Capsules and PTFE Molds with ACLAR™ as cover (Cat. No. 10506 or 10508) work well at 65°C. Widely used because of excellent immunolabeling results. LR White is a polar, monomer polyhydroxylated aromatic acrylic resin (see PTFE Molds).

Sections from LR White appear "bumpy" in contrast to Epon resin sections. It is possible that Epon links covalently to tissue proteins while LR White links more weakly. "LR White appears to act in a manner similar to ice in a freeze-fractured specimen."17 A. Yamamoto and Y. Tashiro, using AFM, found a strong relief surface on LR White sections, 3-4nm on average. Depressions ranged from 6 to 30nm. Protrusions could be seen on the complimentary surface. Epon sections show relatively smooth surfaces.18 Micrographs appear similar to freeze-etched images but scaled to nm instead of µm.

17 Carmichael SW: Down the resolution road: freeze-fracture revisited? Microscopy Today, 95-1, Jan/Feb 1995, p 3

18 Yamamoto A, Tashiro Y: Visualization by an atomic force microscope of the surface of ultra-thin sections of rat kidney and liver cells embedded in LR White. J Histochem Cytochem, 42, 1994, pp 1463-1470

Landmark Paper, Freeze-Fracture
Steere RL: Electron microscopy of structural detail in frozen biological specimens. J Biophys Biochem Cytol, 3, pp 45-60, 1957

Newman GR: Use and abuse of LR White. J. Histochem, 19, 118, 1987.
Newman GR: LR White embedding medium for colloidal gold methods. In: Colloidal Gold: Principles, Methods, and Applications, V. 2, (Hayat MA, ed)

PELCO® Technical Notes, LR White Embedding Medium, Prod. Nos. 18181 & 18182