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Traditional methods
Introduction
The aim of this appendix is to include discussion and methodology from the chapters Lipids, Proteins, Nucleic Acids and Enzyme Histochemistry which were included in previous editions of this book. These technologies have largely been superseded in the modern laboratory but are still used in teaching courses worldwide. For a full discussion and omitted methods please refer to the sixth edition of this text.
Lipids
These methods have an application in diagnostic pathology for nervous system disorders. The myelin sheath is particularly rich in lipids, being composed of a compacted cell membrane which is a lamellar structure of cholesterol and phospholipids. The lipid demonstration methods can aid in the diagnosis of demyelination and the lipid storage disorders.
Lipids may be defined as any one of a group of fats or fat-like substances. These fats include:
-
True fats – esters of fatty acids and glycerol.
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Lipids – phospholipids, cerebrosides and waxes.
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Sterols – cholesterol and ergosterol.
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Hydrocarbons – squalene and carotene.
Classification
Lipids may be classified as a mixed group of substances with the common characteristics of solubility in organic solvents and insolubility in water. They can be organized as simple lipids, compound lipids or derived lipids.
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Simple lipids: esters of fatty acids with alcohols, including fats, oils and waxes. Fats are neutral esters of glycerol with saturated or unsaturated fatty acids. Oils may be similar to fats but are liquid at room temperature. Waxes are esters of higher alcohols with long-chain fatty acids. Simple lipids are usually found in the body as energy stores in adipose tissue. Waxes are usually found in plant and some animal species.
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Compound lipids: usually consist of a fatty acid, an alcohol a phosphoric acid and a nitrogen base. These can be found in the brain and central nervous system.
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Derived lipids: fatty acids which can originate from the simple and compound lipids by means of hydrolysis. Cholesterol, bile acids, sex and adrenocortical hormones are examples.
Fixation and microtomy
The common method for demonstrating tissue lipids is with fresh frozen (cryostat) sections. Some degree of fixation may be necessary so that lipids and the sections themselves are able to withstand the potentially destructive or solvent effects of histochemical reagents. The only reagents which truly fix lipids are osmium tetroxide and chromic acid, but both these greatly alter the chemical reactivity of the lipids. Frozen or cryostat sections are required for lipid histochemistry because routine processing for paraffin wax and resin sections will result in the extraction of all but a few protein-bound lipids from the tissue. Although lipids are not strictly fixed by formaldehyde, they are better retained in a section when the supporting matrix of tissue proteins has been fixed.
Fat stains and the Sudan dyes
Lipids which exist as fats, namely the oily and greasy hydrophobic lipids, have an affinity for the Sudan dyes. For many years a wide range of these compounds has provided almost the sole means of staining lipids.
Oil Red O in dextrin (modified by )
Fixation
Fresh frozen or NBF.
Sections
5 μm mount on slides, air dry.
Solutions
Oil Red O solution
| Oil Red O | 0.5 g |
| Absolute isopropyl alcohol | 100 ml |
Allow to stand overnight.
Dextrin solution
| Dextrin ∗ | 1 g |
| Distilled water | 100 ml |
∗ Bacteriological grade or Type III from Sigma, from corn. Dextrin is hydrolyzed corn starch. VWR Scientific also has small quantities, must be most soluble form of dextrin.
Working solution
| Stock Oil Red O | 60 ml |
| Dextrin | 40 ml |
Mix and allow to stand for a day or more. Stable for months, filter before use.
Method
- 1.
Place slides directly into filtered 0.5% Oil Red O in dextrin. Stain for 20 minutes, rinse with running water briefly.
- 2.
Counterstain with Gill II hematoxylin for 20–30 seconds. Rinse with water, blue, coverslip with aqueous mounting media.
Results
| Lipids | brilliant red |
| Nuclei | blue |
Standard Sudan black B method for fats and phospholipids
Fixation and sections
Cryostat sections post-fixed in formal calcium; short fixed frozen sections; unfixed cryostat sections (preferred).
Method
- 1.
Rinse sections in 70% ethanol.
- 2.
Stain for up to 2 hours in saturated Sudan black B in 70% ethanol.
- 3.
Rinse in 70% ethanol to remove excess surface dye and wash in tap water.
- 4.
Counterstain nuclei with Kernechtrot for 2–5 minutes.
- 5.
Wash well and mount in glycerine jelly.
Results
The standard Sudan black procedure stains unsaturated esters and triglycerides blue-black . Some phospholipids appear gray and those in myelin exhibit a bronze dichroism in polarized light.
Bromination enhances the reaction of these lipids and in addition stains lecithin, free fatty acids and free cholesterol.
Note
The Sudan black solution should not be oversaturated or sections will be covered in a fine deposit. Fixation enhances the staining of phospholipids (present in all tissues) and this is unwanted in general use, thus unfixed sections are preferred.
Cholesterol
Perchloric acid-naphthoquinone (PAN) method for cholesterol ( )
Fixation and sections
Formal calcium fixed frozen section; cryostat sections post-fixed in formal calcium.
Preparation of reagent
| 1:2 naphthoquinone-4-sulfonic acid | 40 mg |
| Ethanol | 20 ml |
| 60% perchloric acid | 10 ml |
| 40% formaldehyde | 1 ml |
| Distilled water | 9 ml |
Mix and use within 24 hours.
Method
- 1.
Air dry sections onto slides.
- 2.
Treat with 1% ferric chloride for 4 hours.
- 3.
Wash well in distilled water.
- 4.
Carefully paint the sections sparingly with the reagent using a soft camel-hair brush. (Note: wash the brush thoroughly with water after each use, and dry.) Heat them on a surface at 70°C for 1 or 2 minutes, until the color develops. The sections are kept moist by gently replenishing the reagent from time to time.
- 5.
Place a drop of perchloric acid on a cover glass and lower section into position.
Results
| Cholesterol and related steroids | blue |
Sphingomyelin
Sodium hydroxide-ferric hematoxylin/DAH method
Fixation and sections
Formalin fixed sections, preferably mounted on chrome gelatin subbed slides.
Method
- 1.
Treat sections with 2 M sodium hydroxide for 1 hour at room temperature.
- 2.
Wash gently but thoroughly in a large volume of water.
- 3.
Rinse in 1% acetic acid for 5 seconds.
- 4.
If section has become detached from the slide, remount it and proceed with the ferric hematoxylin method as described for phospholipids in previous editions of this text ( ).
Results
| Sphingomyelin | blue |
Cerebrosides
Modified PAS reaction for cerebroside ( )
Fixation and sections
Cryostat sections post-fixed in formal calcium; fixed frozen sections.
Preparation of reagent
| 98% formic acid | 45 ml |
| 100 vol hydrogen peroxide | 4.5 ml |
| Concentrated H 2 SO 4 | 0.5 ml |
Prepare an hour before use and stir occasionally with a glass rod inside a fume hood, to release bubbles of gas from the solution.
Method
- 1.
Mount duplicate sections onto separate slides and extract one of these with chloroform methanol (2:1 v/v) for 1 hour at room temperature.
- 2.
Deaminate both sections in 10% aqueous chloramine T for 1 hour at 37°C.
- 3.
Wash slides vigorously and as rapidly as possible, one at a time, in a large volume of water before transferring them immediately to performic acid for 10 minutes. The washing must be swift yet thorough, to avoid swelling and detachment of sections from slides.
- 4.
Wash well in distilled water.
- 5.
Treat with a filtered saturated solution of 2:4 dinitrophenyl hydrazine in 1 M HCl at 4°C for 2 hours.
- 6.
Wash well in water.
- 7.
Treat with 0.5% periodic acid for 10 minutes.
- 8.
Wash in distilled water.
- 9.
Stain in Schiff’s reagent for 15 minutes.
- 10.
Rinse in distilled water and wash in tap water for 15 minutes to develop color.
- 11.
Counterstain nuclei with Mayer’s hematoxylin or Carazzi’s hematoxylin if required.
- 12.
Wash in tap water, distilled water and finally mount sections in glycerine jelly.
Results
| Cerebrosides | magenta |
Indicated by the difference in staining intensity between the two sections.
Sulfatides
Toluidine blue-acetone method for sulfatide ( )
Fixation and sections
Post-fixed cryostat sections; formal calcium fixed frozen sections.
Reagent
0.01% toluidine blue in phosphate-citrate buffer at pH 4.7.
Buffer solution
| 0.2 M Na 2 HPO 4 | 96 ml |
| 0.1 M citric acid | 104 ml |
Method
- 1.
Mount sections onto slides.
- 2.
Stain for 16–18 hours in buffered toluidine blue.
- 3.
Wash in water.
- 4.
Dehydrate with acetone for 5 minutes.
- 5.
Mount in DPX.
Result
| Sulfatide deposits appear metachromatic red, brown or yellow |
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