Introduction 
All tissues received in the EM Lab for routine processing must be fixed in 2% buffered glutaraldehyde. Vials of fixative are provided and kept refrigerated.

All tissues submitted for EM must be properly identified and must include a completed EM Request form (pdf format). A completed Surgical Pathology Accession Sheet can act as a backup with the following information:

• Surgical Number
• Patient Name
• Type of Tissue
• Hospital Submitting Tissue
• MCV - In Patient or Out Patient
• Attending Physician

Three Basic Tissue Preparation Steps
There are 3 basic steps in the tissue preparation process: 1) Fixation 2) Embedding 3) Sectioning.

1) Fixation
To preserve the desired natural state, chemicals are often used to "fix" the structures in place. No fixative is perfect. Structures can be preserved by carefully choosing which fixatives to use. In 1963, Sabatini and Co-workers introduced the combination of glutaraldehyde followed by osmium tetroxide. This combination is now the foundation for most standard fixation procedures used today, and the one which is routinely used in our laboratory.

Glutaraldehyde is a good general fixative for proteins and ground substances, like glycogen. It does not fix lipids very well. The penetration is slow--1mm/hour. The pH range is 7.2-7.4 for most samples.

Osmium tetroxide is a good fixative for lipids and preserves fine detail. It also works as a stain mordant. Exposing tissue to osmium longer than one hour will cause extraction of components. There is no reaction to glycogen. The penetration is very slow--less than 0.5mm/hour.

Size is very important because of the penetration properties of glutaraldehyde and osmium tetroxide. Therefore, tissue pieces must be cut into approximately 1 mm size pieces to optimize fixation. Fixation should be completed before the onset of autolytic changes.

Because ultrastructural components can disintegrate so quickly, it is important to initiate the fixation process as soon as possible. Fixation by immersion should take place within seconds.

Microstructures are easily crushed. Care must be taken not to mechanically traumatize the tissue when cutting it into pieces. Do not pick up tissue samples with tweezers. Use only a double edge razor for cutting.

All tissues are received in the EM Lab fixed in 2% glutaraldehyde in 0.1M phosphate buffer, and are allowed to fix at least two hours at 4 C (preferably overnight). The tissue should already be cut into 1mm-sized pieces.

2) Embedding
Washing
The tissue is washed in phosphate buffer (3 x 15 minutes) and transferred into 1% osmium tetroxide solution for one hour at 4 C. It is then rinsed again in the phosphate buffer.

Dehydrating
The tissue is dehydrated through a graded series of ethanol solutions and propylene oxide (automatic processor LYNX el).

Encapsulating
The tissue pieces are placed into BEEM capsules which are filled with Poly/Bed 812 resin and cured in a 68 C oven overnight. The hardened blocks are brought to room temperature and removed from the capsules. At this point they are ready to be sectioned.

3) Sectioning 
Ultramicrotomy

Thick sections are cut, using a glass knife at a 45 degree angle, one to three-micron-thick (ultramicrotome) from the embedded blocks and placed onto glass slides. The sections are stained with 0.1% toluidine blue and coverslipped, then delivered to the pathologist for review.  

• stained with uranyl acetate and lead citrate
• rinsed and placed into grid holders
• properly labeled. 

The pathologist views the grids in the Philips-400 TEM at an accelerating voltage of 60kV. The film is developed and 8 x 10 photomicrographs are made. These photomicrographs are labeled with appropriate information and delivered to the pathologist for examination.

Alternative Tissue Prep from Paraffin Block
Principle
This procedure is used when resin embedded tissue samples are unavailable or the need for electron microscopy was not anticipated. In certain cases, the retrieval of tissue embedded in paraffin (formaldehyde-fixed), allows for adequate preservation of the ultrastructure for diagnosis.

Procedures
The paraffin block and its paperwork is received from the pathologist, with the area for embedding clearly marked. Ideally, the pathologist has already removed the selected area from the paraffin block and placed it in a small glass vial (provided by the EM Lab).

An EM REQUEST is ordered. The following preparation steps are then undertaken:

1. xylene 15min.
2. xylene 15min.
3. xylene overnight on rotator
4. Tissue pieces are removed from xylene and trimmed, if necessary, with a clean razor blade to 0.5-1mm size pieces for processing. All pieces are placed back in fresh xylene.
5. xylene 15min.
6. xylene 15min.
7. 2%OsO4 in xylene 1 hour (light sensitive, cover)
8. 100% ETOH 10min.
9. 100% ETOH 10min.
10. propylene oxide(PO) 10min.
11. PO 10min.
12. 1:1 PO:Resin 30min. on rotator
13. 3:1 PO:Resin 30min. on rotator
14. 100% Resin 30min. on rotator
15. EMBED

Prep time is 3 hours and 40 minutes. The blocks are ready for thick and thin sectioning the next day.

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