Research Techniques I (Biology 513)

Research Techniques I (Biology 513) Tissue processing

Step 1: Dehydration • Why dehydrate tissue Answer: hydrated tissue is soft and contains hollow spaces (lumen) which deform upon sectioning • To prevent deformation, hollow areas are replaced by a medium (either wax or plastic)

Step 1: Dehydration Procedure : process tissue through a series of graded alcohols. (i.e., 30, 50, 80%) Problem – dehydration causes tissue shrinkage • To minimize shrinkage process tissue through more steps of graded alcohols (i.e., 30, 50, 70, 80, 95%)

Step 1: Dehydration Two factors to consider when dehydrating (i) Time in each alcohol step should be no more than 1 hr. Longer times leads to tissue brittleness (ii) Size counts. The smaller the tissue section, the less time needed in each alcohol step

Step 2: Clearing Why clear the tissue? Answer: alcohol will not mix or dissolve with molten paraffin. Tissue is immersed in some fluid that is miscible with both alcohol and paraffin. Common clearing agents xylene, toluene, you will use histoclear

Step 2: Clearing Problems Clearing agents harden tissue, hence times must be minimal (up to 1 hr) Rapid evaporation, hence stopper bottles

Step 3: Paraffin infiltration • Different types of waxes melt at different temperatures. Range is 50 – 68 °C • For 5 µm sections use a wax with a melting point of 56 – 58 °C

Step 3: Paraffin infiltration Problems • Temperature regulation is critical. Overheating paraffin will destroy some of its properties and reduce sectioning quality • Limit the time tissue stays in contact with hot paraffin (up to 1 hr). Heat causes tissue shrinkage

Step 3: Paraffin infiltration • To aid in paraffin penetration, a warm vacuum oven may be used. (i.e., air trapped in lung tissue is removed in this manner)

Step 4: Embedding Which embedding medium to use? Paraffin wax • Advantage: can section tissue 2 – 15 µm thick • Disadvantage: heat required, tissue shrinkage (25%) Plastics • Advantage: no heat required, minimal shrinkage (< 10%) • Disadvantage: narrower range of section thickness than paraffin (2 – 10 µm)

Step 4: Embedding Procedure (i) Pre-warm all handling instruments. Work as quickly as possible WITHOUT physically damaging the tissue (ii) Before embedding determine the correct orientation of the tissue (iii) Place tissue in fresh paraffin in a container (plastic mold) and then solidify the wax by cooling it

Step 4: Embedding Procedure (iv) A second disposable (plastic) mold must be placed above the original mold, to enable the tissue to be sectioned. ** wax for this second mold must be cured at the same time as the original mold ** (v) Paraffin wax contains 7 – 15% dissolved air. Cooling too slowly or rapidly will lead to uneven pocketing of air molecules, resulting in difficulty in sectioning

Step 5: Sectioning Procedure 1. Trim the block into squares or rectangles to reduced the amount of wax around the tissue 2. Make certain the microtome is set to cut 5 µm sections

Step 5: Sectioning Procedure • When cutting the tissue orient the tissue as shown below

Step 5: Sectioning Procedure 4. Make certain the knife angle tilt is 4° a. Knife will compress section b. Knife will act like a chisel and gouge section. 5. Samples should be placed in the freezer at least 30 min. prior to sectioning.

Step 5: Sectioning Tissue ribbons

Step 6: Mounting sections • The placing of sections onto glass slides Procedure 1. Clean slides with 95% ethyl alcohol 2. BEFORE placing sections onto slides label the slide on the frosted side. Use a pencil only (will not wash off later) 3. After cutting the sections, use a heated (45 °C) water bath (the technique will be demonstrated) to place the sections onto the slides and allow them to dry overnight. Keep free from dust 4. Heat fix sections onto slides (65 °C for 30 min.). Allow to cool

Research Techniques I (Biology 513)