Processing Reagents EXAM 4

Processing purpose

Fixed tissues are taken from an aq state to a nonaq state → support to withstand microtomy

Tissues can shrink 20% or more

Processing steps

Fixation, dehydration, clearing, infiltration

Dehydration step

Alcohol removes free water

Clearing step

Xylene removes alcohol from tissues (bc alcohol and paraffin are not miscible)

Removes the dehydrant agent. Must be miscible with infiltration media

Infiltration step

Paraffin provides mechanical support to tissue

Excessive dehydration results

Brittle/hard tissue and morphological damage or distortion

Chatter

Under dehydration results

Soft and mushy tissue that sections poorly w.absent or patchy staining.

Paraffin will not penetrate tissue containing water

Using alcohol as a dehydrating agent

Removes water by gradually increasing [ ] at each station. Gradually reduces morphological damage. Last station has 0 H2O to avoid carryover

Hydrometer

Measures alcohol concentration

When using a phosphate buffered fixative…

1st alcohol station must be at least 70% to avoid salt precipitation.

With inadequately fixed tissues…

Alcohol will be both a fixative and dehydrant

Clearing agents

Aromatic hydrocarbons: xylene, toluene, benzene.

Chloroform, xylene substitutes (limonene, aliphatic hydrocarbons), Universal solvents (acetone, dioxane, tert butanol, tetrahydrofuran)

Universal solvents

Dehydrate and clear in 1 step.

Miscible with both water and paraffin.

NOT for delicate tissues (bc of cellular distortion)

What are the universal solvents?

Isopropanol, acetone, tert butanol isomer, dioxane, tetrahydrofuran

Beeswax and paraffin

incr stickiness and adhesion, soften paraffin

Rubber and paraffin

increase elasticity, decrease brittleness, softens p.

Plastics/polymers and paraffin

increase hardness and support

DMSO and paraffin

accelerate tissue infiltration

Paraffin w/high melting point

More plastic, harder paraffin, better support for hard tissues, thin sections easier but microtomy harder

Paraffin w/LOW mp

More wax, softer paraffin, less support for hard tissues. Thin sections hard but microtomy easier

Plastic point

Temp where hardening/crystallization occurs.

As plastic increase, so does plastic point

Large crystals =

high MP and PP, harder paraffin

Smaller crystals =

Low MP and PP, better support and easier microtomy

Molten paraffin at..

2-4 above melting point

Paraffin on tissue processor should be at…

57-62 bc melting is 55-58

Overheating of paraffin causes

brk down of paraffin molecule → poor support and overhardening/distortion of tissueWh

When to use alternative infiltration media

for lipids, enzymes, examination of ultrastructure, undecal spec

EX: agar/gelatin, 30% sucrose, carbowax, celloidin, resins, epoxy resin

Agar/Gelatin

Used for small fixed fiable pieces (not for lipids, enzymes)

2 embedding mediums = double embedding

30% Sucrose

Used to obtain frozen sections on formalin fixed tissue.

Acts as cryoprotectant, high quality frozens, staining is easily performed bc tissue has already been fixed.

After solution is used, it is then embedded into OCT for frozens.

NOT for embedding, only infiltration

Carbowax

Only for special projects → Lipids and enzymes.

WATER SOLUBLE

Dehydration and clearing not necessary, but tissue sections will dissolve on routine water bath. → Preserves lipid and enzymes.

Will not infiltrate tissue containing large amts of fat or CNS tissues

Celloidin

For dense or hard tissues.

Hazardous.

Very slow process required. NEEDS SLIDING MICROTOME.

Resins

Electron microtomy or undecal bones.

Acrylic resin (methacrylate) was original resin for e- micro., Epoxy resin is newer

Acrylic resin (methacrylate)

Original for e- micro.

LR White and LR Gold for e- micro, Light micro

Methyl (MMA) for undecal

Epoxy Resin

New for EM

Transitional solution = propylene oxide

EX: Araldite, Epon, Spurr