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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 agentsxylene, 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

+Step 4: Embedding

+Step 5: Sectioning

+Step 5: Sectioning

+Step 5: Sectioning

+Step 5: Sectioning

+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