Freeze Substitution

This method has been used successfully in our laboratory since 1994. The protocol assumes all users will be familiar with routine TEM preparation methods. An important application of this method was made by Dr. A. Whitney who used it to examine MDCK cells grown on filters.

Aldehyde Fixation

Fix cells or tissues in buffered aldehyde solution. We use either 4% formaldehyde (made up from paraformaldehyde) or a mixture of 4% formaldehyde and 0.2% gluteraldeyhde, buffered in 100mM phosphate buffer. Fixation times vary but routinely we use 1 hr. Whole organs are best fixed by perfusiing the with fixative.

Cryoprotection

Incubate fixed material in 2.3M sucrose. This cryoprotection method is also used for material that is to be frozen for cryosectioning. Small pieces of the material are left for 1 hour to infiltrate.

Freezing

Mount the small, cryoprotected sample pieces onto metal pins and immerse in liquid nitrogen. The pins are useful for manipulating the frozen samples during the early stages of the protocol and ensure the sample is submerged during freezing.

Substitution

Quickly transfer the samples into glass vials of methanol on a bed of dry ice in a styrofoam box. Alternatively, the vials can be left in a REVCO freezer. Seal vials well and leave for 8 - 48 hours. Smaller samples will substitute faster than larger samples. Increased contrast can be achieved if 1% uranyl acetate or osmium tetroxide is added to the substitution mix. These heavy metals do not interfere with resin polymerization. Dry acetone can be used as a substitution medium instead of methanol. However, it is important for the acetone to be dry.

Washing

Wash in fresh changes of substitution medium prior to embedding in resin. Usually 2 - 3 changes over a few hours is sufficient.

Embedding in resin (using Lowicryl HM20)

	Time	MeOH:resin
	1 hr.	1:1
	1 hr.	1:2
	1 hr.	pure resin
	overnight.	pure resin

Resin polymerization

Remove sample pieces from the glass vials and place in precooled BEEM capsules or small Eppendorf tubes containing fresh HM20 Lowicryl resin. Keep the tubes on dry ice and seal them as soon as the sample pieces have been added. Place only one sample per tube. Arrange the tubes on a rack in a styrofoam box, covered on the inside with aluminum foil, and pack with dry ice. Cover the tubes with a triangular shaped cardboard box that has been covered with aluminum foil. Place a UV light above the box and leave for up to 3 days to polymerize. The resin polymerizes in indirect 360 nm UV light. Too rapid polymerization will result in shrinkage of the block and uneven polymerization. Once the blocks have hardened, polymerization can continue at room teperatures using direct sunlight or the UV light used to sterilize cell culture hoods

Sectioning

Lowicryl resins do not crosslink well at tissue-resin borders. It is important, when trimming the block, to make sure the sample is not dislodged. The best way to trim the block is by using either a glass knife or one of the trimming devices currently available (supplied by Diatome US or Harris Diamond Co.). Once the block is trimmed, it is fairly easy to section using routine sectioning methods. Diamond knives are recommended. The HM20 resin will not change shape if water is splashed onto the block. Sections can be picked up from the water surface with metal specimen grids. The sections are now ready to be labeled with affinity markers