TCA

4-8 days very slow acting Good nuclear staining Weak

Sulfurous

Weak For minute pieces of bone

Chromic Acid (Flemmings fluid)

Chromic acid, osmium tetroxide, glacial HAc Fixative and decalcifier Nuclear staining with hematoxylin is inhibited Forms precipitate at the bottom *carcinogenic, corrosive to skin

Citric Acid citrate buffer pH4.5

With zinc sulfate and chloroform 6 days slow Good nuclear and cytoplasmic staining

HNO3 - summary

Most common, rapid (1-3 days) Yellow color imparted prevented by 5% sodium sulfate, wash in running water x 12 hours or + 0.1% urea Formol Nitric (1-3 days) Perenyis w/ chromic acid + abs. EtOH

Decalcifies & softens; slow (2-7 days)

Phloroglucin Nitric most rapid (12-24h); poor nuclear staining

HCl slower than HNO3 Formic fixative/decalcifier, slow

Formic Acid-Sodium Citrate solution - slow

TCA good nuclear staining, no washing out; weak & slow acting Sulfurous very weak Chromic (Flemmings) fixative/decalcifier Citric Acid Citrate buffer pH4.5 - slow Von Ebners NaCl, HCl

Chelating Agents

EDTA (versene) Excellent staining Very slow Slight tissue hardening produced

Ion Exchange Resins

* artifacts produced, usually caused by CO2 bubbles slow

Electrophoresis (Electrical ionization)

Formic acid, conc HCl, distilled water

Tests for Completeness of Decal:

Physical / Mechanical X ray / Radiological Chemical litmus paper red if due to acidity, add NH3 drop by drop blue litumus; if cloudy still w/ calcium; if clear: +ammonium oxalate, 30 mins cloudy if incomplete

Tissue softeners

Perenyis 12 24 hours 4% aqueous phenol 1 3 days Molliflex (swollen & soapy appearance) 2 % HCl 1 % HCl in 70 % alcohol

*Post decalcification

Remove acid by saturated lithium carbonate solution or 5-10 % aqueous NaHCO3 for several hours Running tap water If EDTA is used use 70 % alcohol

*Rate of decalcification

More concentrated acid solutions more rapid but more harmful to tissue Heat hastens decalcification, but increases damaging effect of acids to tissues Mechanical agitation, sonication Ideal time 24 to 48 hours

Dehydration most critical stage of processing difficult to correct

Ideal Dehydrating Solution:

Rapid acting w/ no tissue shrinkage or distortion Should not evaporate fast Can dehydrate even fatty tissues Non toxic Not fire hazard Should not harden tissues excessively

Dehydrating Agents:

Alcohol most common Acetone Dioxane Cellosolve Triethyl phosphate THF

Alcohol progressively increasing

Ethyl best, fast, mixes with water and penetrates easily, not poisonous, cheap Methyl toxic, for blood films Butyl slow, for Plant and Animal microtechnique

* Alcohol

Prolonged storage in 70 % alcohol macerates tissues Directly placed in high grade alcohol shrinkage & hardening of tissues

Acetone

Cheap Rapid acting Volatile Tissue shrinkage

Dioxane (Diethylene Dioxide)

Excellent dehydrating & clearing agent Less tissue shrinkage Tissues can be left here for a long time Ribbons poorly Expensive Dangerous vapor cumulative Graupners method (dioxane, paraffin wax) Weisebergers method (gauze, Ca oxide)

Cellosolve

Ethylene Glycol Monoethyl Ether Rapid Storage without producing hardening or distortion

Triethyl Phosphate

Minimum shrinkage Minimum distortion and hardening of tissue

Tetrahydrofuran - THF

Dehydrates and clears Less shrinkage Easier cutting w/ fewer artifacts Non toxic; 6 months exposure conjunctival irritation Offensive odor (use well ventilated room)

*additives to dehydrating agents

4 % phenol added to 95 % ethanol softens hard tissues Hard tissues immerse in glycerol/alcohol mixture or in Molliflex

Clearing / Dealcoholization

Substance will dissolve wax with which the tissue is to be impregnated After staining transparent tissues

High refractive index

Clearing Agents:

Miscible with alcohol and paraffin &/or mounting medium Should not produce excessive tissue shrinkage & hardening Should not evaporate quickly in a waterbath Should make tissues transparent

Clearing Agents:

Xylene Toluene Benzene Chloroform Cedarwood oil Aniline oil Clove oil CCl4

* Clearing Agents (cont)

* methyl benzoate and methyl salicylate

slow Dehydrates and clears Non toxic Offensive odor

`THF

Xylene (Xylol)

Most common, colorless Most rapid Cheap Milky if incomplete dehydration Hardens/shrinks tissues not for nervous system & lymph nodes Hard/brittle tissues if > 3 hours

Toluene

Not carcinogenic; but will emit fumes that are toxic upon prolonged exposure Rapid acting Tissues do not become excessively hard and brittle even if left here for 24 hours Slower than xylene & benzene expensive

Benzene

Rapid acting Volatilizes rapidly in paraffin oven Tissue shrinkage if left for a long time Carcinogenic; aplastic anemia