UNIT IX – IMPREGNATAION AND EMBEDDING Impregnation/Infiltration Removal of clearing agent from tissue Replacement by a medium to fill all tissue cavities To give a firm consistency to specimen To allow easier handling and cutting of suitably thin sections Prevent distortion Prevent alteration of spatial relationships of tissue and cellular elements EMBEDDING/ CASTING/BLOCKING Placement of impregnated tissue into a precisely arranged position in a mold with a medium Allowing medium to solidify Allowing specimen to be handled and fixed to the microtome block without damage to the actual tissue EMBEDDING MEDIUM Paraffin, Celloidin, Gelatin GOOD EMBEDDING MEDIUM -Easily converted from liquid to solid after infiltration -Solidification (Crystallization, Hbonding, covalent bonding/ polymerization) -Good cutting qualities (Homogeneity, hardness, plasticity, elasticity) -Cheap -Readily available

Paraffin Celloidin/ coloidion

Gelatin

Crude petroleum Simplest Most common Best impreg+embed Routinely used MP: 45, 54, 56, 58C Most MP : 55-60C

Purified nitrocellulose, for large hollow cavities spn, hard dense tissues (bone, teeth) -Thin2% -medium4% -thick8%

-Rarely used -to avoid dehydration -for histochemical and enzyme studies -doesn’t require dehydration and clearing

Advantage Rapid, 24hr For processing neurological tissue

Dis-advantage

Not recommended for fatty tissue

-Slow (days-weeks) -Thin are difficult to cut -very volatile

Method 1. Manual 2. Automatic (autotechnicon) 3. Vacuum embedding (negative atm pressure inside emb.oven to hasten removal of air bubbles and clearing agent from tissue block)

1. Wet (bones, teeth, large brain section, whole organ) 70-80%Alcohol avoid dehydration and shrinkage 2. Dry (whole eye) Gilson’s mixture = chloroform + cedarwood oil

Factors -Nature & tissue size -Clearing agent (Benzene, xylol = easily removed) (Chloroform & cedarwood oil = difficult to remove)

Precaution Wax may be melted and reused (only 2x)

Substitute for Paraffin

1. Paraplast (56-57C) Dimethyl sulfoxide/ DMSO, high dipole moment to carry

paraffin to tissue.

-more elastic, resilient

-expensive

2. Embeddol (56-58C) less brittle, compressible

3. Bioloid (eyes)

4. Tissue mat (rubber)

5. Ester Wax (46-48C) Sliding/sledge microtome, relative hardness of wax

6. Water soluble wax (Carbowax) 38-42 or 45-46 DOUBLE EMBEDDING METHOD (2 media) -infiltration with celloidin then embedding with paraffin mass. -For dense firm tissue like brain

-Leukhart’s embedding mold = 2 L-shaped strips of heavy brass -Compound embedding unit -Plastic embedding rings and base molds = tissue tek -disposable embedding mold = peel away, plastic ice tray, paper boats Trimming of blocks : remove excess paraffin on both sides of block for attachment to microtome block holder.