+ 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

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+ Step 5: Sectioning

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+ 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