Sample Preparation There are multiple ways that tissue speci

Sample Preparation: There are multiple ways that tissue specimens can be prepared for electron microscopy. Your book has some samples of techniques for varying types of samples. However, we would like to at protocols (the method of completing the process from acquisition to for a few different samples. for the following tissue: Spleen, Liver, Kidney, Your assignment is the following: create protocols and subcutaneous tissue), skeletal muscle, skin (a sectional representation containing both cutaneious and cartilage. appropriate, however in creating these for protocols can include any method you would think of the tissue simply protocols, keep in mind that the purpose is to complete a general examination initial observational purposes.

Solution

Answer:

The longitudinal midline and the transversal line at the lumbar sacro point of the dorsal skin are incised and the skin is turned over to expose the subcutaneous side of the skin.

1. FIXATION:

Tissue can be fixed by immersion or perfusion. The most commonly used method is immersion. Fixation time is variable, depending on tissue, but usually from 4 hours to overnight at 4 degrees (refrigerator).

a. Primary Fixative: 1/2 strength Karnovsky’s.

2% Paraformaldehyde/2.5% Glutaraldehyde

This is buffered with a 0.2M Cacodylate Buffer

b. 0.1M Cacodylate Buffer wash

c. Post Fixative: 2% aqueous OsO4/0.2 M Cacodylate Buffer (A 1:1 solution for 2 hours in the refrigerator)

d. 0.1 M Cacodylate Buffer wash

2. DEHYDRATION:

Dehydration with ethanol:

35% ETOH 15 min. and decant (optional)

50% ETOH 15 min. and decant

70% ETOH 15 min. and decant

95% ETOH 15 min. and decant

100% ETOH 15 min. and decant

100% ETOH 15 min. and decant

Propylene Oxide 15 min. and decant

Propylene Oxide 15 min. and decant

1:1 PO/Epon resin overnight

3. EMBEDDING:

Embedding with Epon:

Next morning, change out to fresh Epon 812 for 1-3 hours.

Embed (always) in fresh Epon 812

Polyethylene capsules are placed in a holder and numbered strips of paper are inserted.

A drop of fresh Epon 812 is placed in the capsules and the specimen is transferred to the appropriate capsule.

The “blocks” are cured for 48 hours in a 60 degree oven.

4. SECTIONING:

1 micron or thick sections are taken to find the area of interest

Take ultra thin sections (~70nm) and place on grids

5. POST STAINING:

Stain grids in uranyl acetate for 2 hrs.

Stain grids in lead citrate for 5 min.