USE OF ULTRASTRUCTURAL HISTOCHEMISTRY

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44

USE OF ULTRASTRUCTURAL HISTOCHEMISTRY

Barbara A. Nichols

I. GENERAL INTRODUCTION

Cytochemical methods for electron microscopy have the unique capability of ultrastructurally localizing enzymes or chemical substances. There are several requirements for success with these methods. It is essential that the enzyme be fixed in its native position in the tissues, yet retain sufficient activity to be demonstrable by the cytochemical technique subsequently used. It is essential that the sub- strate have access to the enzyme, and that the product which results from the cytochemical reaction be electron dense and remain at the site of its formation. Although there are fewer cytochemical methods available for electron microscopy than for light microscopy, there are nonetheless several volumes now devoted to the discussion of them (1).

This chapter is intended to provide some practical infor- mation about the localization in mononuclear phagocytes of peroxidase and two acid hydrolases, acid phosphatase and aryl

MONONUCLEAR PHAGOCYTES 4 1 3 All rights of reproduction in any form reserved.

ISBN 0-12-044220-5

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414 METHODS FOR STUDYING MONONUCLEAR PHAGOCYTES sulfatase, by methods that have been used successfully in our laboratory (2 - 5 ) . These methods have proven applicable to all mammalian species thus far studied.

A. General Considerations

Enzymes are easily inactivated by compounds such as osmium that are routinely used in preparing tissue for electron microscopy. Because osmium vapors are very pervasive, the histochemistry area of the laboratory must be completely separated from rooms where osmium is used. It is also essential that the glassware used in cytochemistry never be used in other parts of the laboratory because of possible contamination by enzyme- inactivating substances. Careful dish washing is also a basic requirement of the well-run histochemistry laboratory. After washing in Tetrox detergent, glassware should be rinsed 7 times in tap water, followed by 7 rinses in double-distilled water.

B. Fixatives

Glutaraldehyde is the fixative of choice for most cytochemical procedures (6), although brief (5-min) fixation with dilute Karnovsky's fixative (7) may also be used if the enzyme to be studied is relatively stable to fixation. The purity of the glutaraldehyde is fundamental to the success of a cytochemical experiment, since impurities lead to the inactivation of enzymes (6, 8 ) . The purity of commercially available glutaraldehyde greatly varies (9). Glutaraldehyde from Electron Microscopy Sciences has been used with success in our laboratory, although that from other suppliers may be equally good.

Before glutaraldehyde is used, its purity should be established by ultraviolet spectroscopy (8 - 1 0 ) .

C. Fixation

One of the most difficult steps in cytochemical work is the selection of a method of tissue fixation that adequately pre- serves both tissue structure and enzymatic activity. In general, fixation must be brief if adequate amounts of enzyme are to be preserved for cytochemical demonstration. However, tissue that has been only minimally fixed may lose structural in- tegrity during subsequent preparation. This is particularly true when the enzyme to be demonstrated has a pH optimum in the acid or alkaline range. If intracellular membranes are disrupted as a result of inadequate cellular preservation, it is

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V. MORPHOLOGY OF MONONUCLEAR PHAGOCYTES 415 clearly impossible to define the intracellular localization of an enzyme with confidence. The best conditions of fixation must be determined by trial and error for each tissue and each enzyme, since enzymes differ in their susceptibility to inactivation by fixation. Optimally, biochemical analyses can be used to determine the amounts of enzyme in tissue before and after various fixation treatments (11-13). As an additional cautionary note, it is highly desirable when carrying out cytochemical experiments to fix parallel samples of tissue with formaldehyde - glutaraldehyde for optimal morphological preservation (without cytochemical incubation) to ensure that the structural relationships observed intracellularly in the incubated specimens have not been altered by the conditions of the cytochemical test.

Ό. Collection and Handling of Tissues

Tissue is fixed in situ or as soon as possible after re- moval from the experimental animal. For example, fixative is used to wash cells from the lungs or peritoneal cavities as previously described (4). To separate leukocytes from red cells as rapidly as possible, Kaplow buffy coat tubes (The Virtis Company, Inc., Gardiner, New York 12525) are routinely used in our laboratory (14). Blood is drawn into a heparin- ized syringe (50 U heparin/ml of blood) and immediately dis- pensed into Kaplow tubes. The tubes are then centrifuged in a table-top centrifuge (Sorvall GLC-2) with a swinging bucket head at top speed for 10 min, which gives a good separation of the buffy coat. The serum is drawn off and discarded. The buffy coat is then drawn off and immediately dispersed in glutaraldehyde fixative.

Cell suspensions (i.e., bone marrow, blood leukocytes, alveolar or peritoneal macrophages) are centrifuged and resus- pended between changes of solution until postfixâtion in osmium. Prior to osmication, the cell suspensions are pelleted in a Beckman Microfuge. The resulting mass of cells is co- herent and can be cut from the 0.4-ml plastic centrifuge tube,

chopped into 1/2-mm cubes, and handled subsequently as tissue blocks.

Solid tissues, such as lung or liver, must be cut after fixation into 20- to 40-ym sections with a Smith-Farquhar Tissue Chopper (Sorvall Instruments) (10) or an Oxford Vibra- tome. Chopped sections can then be transferred manually through changes of solutions, or solutions can be decanted from the sections.

When possible, the entire cytochemical procedure, from fixation to embedding, should be carried out in one day to minimize loss of enzymatic activity or loss of reaction product,

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TABLE I. Reagents Used in Electron Microscopic Cytochemistry Reagents

3-Amino-l,2 ,4-triazole Ammonium sulfide (20%

solution)

Barbital

Cytidine monophosphate

3, 3'-Diaminobenzidine tetrahydrochloride

Glutaraldehyde (10% and 25%

sol utions)

3-grl yce ro phosphate

grade I

Source*

Sigma Mallinckrodt

Sigma

Electron Microscopy

Sciences Sigma

Use

Inhibitor of catalase Conversion of aryl sul-

fatase reaction pro- duct to lead sulfide

Acetate Veronal wash buffer

Sigma Acid phosphatase sub- strate

Peroxidase medium

Primary fixation

Acid phosphatase sub- strate

Comments

Store desiccated below 0°C Stable at room temperature.

Evolves hydrogen sulfide, a poisonous, noxious gas.

Use only in fume hood.

Stable at room temperature

Store in dark, desiccated below 0°C

Carcinogenic. Avoid inhala- tion of powder and skin contact. Store in dark, desiccated below 0°C Avoid inhalation of vapors

or skin contact

Stable at room temperature

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Table I (Cont'd.)

Horseradish peroxidase

Hydrogen peroxide

p-Nitrocatechol sul- fate

Worthington Tracer of pinocytosis Biochemical

Corp.

Mallinckrodt Peroxidase substrate

Sigma Aryl sulfatase sub- strate

Refrigerate desiccated

Unstable. Use a fresh bottle for each experiment.

Stores well desiccated below 0°C

p-Nitrophenyl phosphate Sigma Acid phosphatase sub-

strate Stable at room temperature.

Osmium (4% solution)

Potassium cyanide

Sodium cacodylate h y dro chl ori de Sodium fluoride

Electron Microscopy

Sciences

Postfixât ion

Mallinckrodt Inhibitor of peroxi- dase

Sigma

Fisher

Wash buffer

Inhibitor of acid phosphatase

Extremely toxic. Avoid in- halation of vapors and skin contact. Use only in fume hood.

DangerJ May be fatal if swallowed. Contact with acid liberates poisonous gas.

Arsenical, carcinogen. Avoid inhalation or skin contact.

Poison. May be fatal if swal-

lowed. Keep bottle in plas-

tic bag to avoid contamina-

tion of other reagents.

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Table I (Cont'd.) Reagents Sodium tartrate

Trizma (Tris) base buffer

Trizma (Tris) maleate buffer

Source Sigma

Spurr's Embedding medium Ladd

Sigma

Use

Inhibitor of some acid phosphatases

Embedding of peroxidase specimens

Peroxidase medium

Acid phosphatase medium

Comments

Stable at room temperature.

Some ingredients reportedly are carcinogenic. Avoid skin contact. Mix in fume hood.

Store refrigerated

Very hygroscopic. Store desiccated, and refrigerated.

Certain pH electrodes do not give accurate results with Tris-maleate buffers. See Sigma Technical Bulletin

Ü106B dated after 1978.

Addresses of chemical companies are given in Appendix.

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V. MORPHOLOGY OF MONONUCLEAR PHAGOCYTES 419

E. Reagents

Many chemicals used in electron microscopic cytochemistry are health hazards (Table I ) . Technicians using these reagents must be informed that they are dangerous so that the appro- priate care can be used in handling them. To avoid skin contamination, laboratory assistants should always wear gloves when washing glassware that has contained these chemicals.

II. CYTOCHEMICAL DEMONSTRATION OF PEROXIDASE

Cytochemical tests for peroxidase have been used extensively in the study of mononuclear phagocytes, since the enzyme myeloperoxidase is endogenous to the monocytes of some mammalian species, and since peroxidatic activity is present in the rough endoplasmic reticulum of many tissue macrophages. For a general review of these topics, consult van Furth (15). In addition, exogenous peroxidase has been used as an extracellular tracer to follow fluid uptake by pinocytosis (16). The same cytochemical incubation procedures are used to demonstrate peroxidase whether the enzyme is endogenous or exogenous.

The fixation of tissues to be incubated for peroxidase may be very brief, or may even be omitted entirely (17, 1 8 ) , since the incubation is carried out at a near neutral pH, causing little tissue damage.

A. The Protocol for Peroxidase (see Appendix for formulas of solutions)

(1) Fix tissues for 10 min in 1.5% glutaraldehyde.

(2) Wash 3 times in sodium cacodylate - HC1 wash buffer.

(3) Incubate in peroxidase medium at room temperature on Dubnoff shaker for 15-60 min.

(4) Wash 3 times in Michaelis wash buffer.

(5) Pellet loose cells in a Beckman Microfuge.

(6) Postfix in Palade's osmium for 1 hr at 4°C.

(7) Stain one specimen in Kellenberger's uranyl acetate stain for 10 min at room temperature.

(8) Omit the stain for an identical specimen.

(9) Dehydrate rapidly in ethanol only, according to the procedure given in the tabulation below:

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420 METHODS FOR STUDYING MONONUCLEAR PHAGOCYTES

70% ethanol 95% ethanol

100% ethanol 3 changes 1:1 ethanol: S purr's medium

2 changes 100% S purr's 2 changes

2 min 2 min 15 min each 20 min each

30 min each or longer

(10) Embed in fresh Spurr's medium.

(11) Cure overnight at 60°C.

B. Technical Problems

There are several technical problems associated with the demonstration of peroxidase. This enzyme is particularly vulnerable to loss of activity, which usually occurs at two points during the procedure. First, there can be loss of activity before incubation, either because of overfixation or because the briefly fixed tissues have been held overnight in buffer. Second, reaction product, once formed, can be removed

from the tissues by subsequent steps of tissue processing.

The reaction is diminished by uranyl acetate staining in block.

Kellenberger's stain is therefore omitted, or two samples are run in parallel, one from which Kellenberger's is omitted and the other in which only a 10-min stain is used. The two samples can then be compared for the best preservation of the tissue and the reaction product. Diaminobenzidine (DAB) reaction product can also be removed by prolonged exposure to sol- vents. For this reason, Spurr's low-viscosity embedding medium (19) is used because dehydration can be carried out

rapidly with the omission of propylene oxide. Rapid polymeri- zation of Spurr's plastic is also an advantage because the sol- vent effects of liquid plastics are reduced. It should be noted, however, that this embedding material reacts with water to produce a brittle plastic, which is difficult to section.

In handling it, care should be taken to avoid moisture, as with other embedding materials.

DAB penetrates tissues slowly. It is advisable to examine unstained 1-ym sections by light microscopy to determine the depth of reactive tissue before selecting an area for thin sectioning.

As a general rule for all cytochemical specimens, an unstained grid is examined by electron microscopy and compared with a stained grid to determine whether reaction product has been removed by the stain on grid. Remarkably enough, reaction product for peroxidase is removed from the plastic sections by uranyl acetate staining on grid. This poses a dilemma, since the omission of uranyl acetate both in block and on grid greatly diminishes specimen contrast, and thus makes the specimen

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V. MORPHOLOGY OF MONONUCLEAR PHAGOCYTES

421 very difficult to examine and photograph. A further complica- tion is that Spurr's plastic does not stain readily with lead stains. It is essential, however, to examine a specimen not stained with uranyl acetate to ensure that no intracellular sites of peroxidase localization are overlooked. Later, a dip of uranyl acetate before 30 min of Reynold's lead citrate may be tried.

C. Interpretation of Results

As with other cytochemical procedures, tests for peroxidase are not specific (20). Other heme proteins, such as hemoglobin and cytochromes, can also oxidize DAB to give a positive result.

Definite identification of a given enzyme must therefore be ob- tained by biochemical procedures.

Considerable study has been devoted to analyzing the condi- tions that favor the demonstration of peroxidase over that of catalase, and vice-versa. Several excellent review articles thoroughly discuss this subject (17, 20 - 23).

Diffusion of reaction product occasionally poses a problem in peroxidase cytochemistry. It is possible that the brief fixation procedures employed in cytochemistry sometimes do not adequately stabilize cellular membranes, so that leakage of reaction product occurs. Generally speaking, the presence of reaction product outside of a membrane-limited organelle should be considered questionable.

D. Control Specimens

Incubations without substrate must be carried out as a

control for the cytochemical tests to ensure that electron-

dense components of the medium are not nonspecifically adsorbed

to cellular structures, leading to false positive results. In

control experiments in which hydrogen peroxide, the substrate

for peroxidase, is omitted from the incubation medium, some

staining may result from the presence of hydrogen peroxide in

the tissue itself. This staining can be eliminated by the use

of catalase in the control incubation medium (20). Aminotria-

zole, once considered a specific inhibitor of catalase, also

inhibits some peroxidatic activities (22). Although potassium

cyanide has no effect on the peroxidase of eosinophils, it is

an effective inhibitor of other peroxidases, including the

peroxidase of other leukocytes. The use of inhibitors is dis-

cussed fully by Essner (20).

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422 M E T H O D S FOR STUDYING M O N O N U C L E A R P H A G O C Y T E S

III. CYTOCHEMICAL LOCALIZATION OF ACID PHOSPHATASE AND ARYL SULFATASE

Lysosomes cannot be identified solely on the basis of their ultrastructural appearance, for they are remarkably heterogene- ous in size, shape, and content. Therefore, their identification in tissue sections depends upon the use of cytochemical methods. Acid phosphatase is the most widely used "marker"

enzyme for cytochemical staining of lysozomes, and the assump- tion is generally m a d e , and appears valid ( 2 4 ) , that a dense body containing acid phosphatase probably contains other acid hydrolases as w e l l . However, it is highly desirable to corro- borate the localization of one enzyme with that of another enzyme in the same cells (25), since cytochemical methods lack specificity. For example, there are not only multiple acid phosphatase enzymes (with possibly different functions) in the same cell (26, 2 7 ) , but also many other phosphatases that have overlapping enzymatic activities (28 - 3 3 ) . Of several techniques available for the demonstration of lysosomal enzymes, tests for acid phosphatase and aryl sulfatase have proved to be the most useful in our hands in the study of mononuclear phagocytes (34).

A. The Protocol for Acid Phosphatase (see Appendix for for- mulas of solutions)

(2) Wash 3 times in sodium cacodylate - HCl wash buffer.

(3) Incubate in acid phosphatase medium on a Dubnoff shaker for 30 - 90 min at 30° - 37°C.

(4) Wash 5-10 times in Michaelis wash buffer.

(6) Postfix in Palade's osmium at 4°C for 1 h r .

(7) Stain in Kellenberger's uranyl acetate for 15 - 60 min at room temperature·

(8) Dehydrate according to the schedule given in the following tabulation:

70% ethanol 5 min 95% ethanol 2 changes 5 min each 100% ethanol 3 changes 10 min each Propylene oxide 2 changes 15 min each 1:1 Epon : propylene oxide 2-4 hr

(9) Infiltrate in pure Epon overnight at 4°C.

(10) Embed in fresh Epon.

(11) Cure at 60°C for 24 h r .

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V. MORPHOLOGY OF MONONUCLEAR PHAGOCYTES 423

B. The Protocol for Aryl Sulfatase (see Appendix for formulas

of solutions)

(2) Wash 3 times in sodium cacodylate wash buffer.

(3) Incubate specimens in aryl sulfatase medium at 30° - 37°C for 30-90 min in a Dubnoff shaking incubator.

(4) Wash until clear with Michaelis wash buffer ( 5 - 7 rinses).

(5) Convert reaction product with 2% ammonium sulfide in Michaelis buffer for 5 - 1 5 min. [in this step, the lead sulfate formed as a result of the reaction is converted to lead sulfide (35). The resulting specimen is easier to section than specimens prepared without this step.]

(6) Wash until clear with Michaelis buffer ( 8 - 1 0 washes).

(8) Postfix in Palade's osmium at 4°C for 1 hr.

(9) Stain in block for 10 - 60 min with Kellenberger's uranyl acetate stain at room temperature.

(10) Dehydrate in ethanol and propylene oxide (see Section III.A), as for acid phosphatase.

(11) Embed in Epon.

(12) Cure at 60°C for 24 hr.

C. Technical Problems

One of the chief technical problems in the cytochemistry of acid phosphatase and aryl sulfatase is the tissue damage that occurs during the incubation of the tissues in the cytochemical medium at an acid pH (5.0 - 5.5). Even though this problem can be anticipated, some experiments must be carried out with only very brief fixation (5-10 min) in order to ob- tain maximal amounts of enzyme deposits. Once the sites of reactivity are determined, slightly longer fixation times can be tried (10-30 min), with the aim of providing better tissue preservation.

Low levels of nonspecific "background" staining in the tissues often make the specimens appear "dirty." Specimens can be "cleaned up" with Kellenberger¹s in block stain (36, 3 7 ) , but it must be recognized that some reaction product is removed, along with the nonspecific staining, during this process. Reaction products composed of lead salts are also removed to some extent by other acid solutions during tissue processing (28). The problem of scanty reaction product may be somewhat diminished by reducing both the time of postfixa- tion in osmium and the time of Kellenberger staining«,

Histochemical latency is one of the most difficult problems to deal with. This property of lysosomes has been

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observed and analyzed extensively in cytochemical investiga- tions (12, 25, 40) of leukocytes, in which granules known to contain lysosomal enzymes were found to remain unreactive for those enzymes after cytochemical tests using lead salt tech- niques. The granules must be disrupted using methods such as freezing or thawing, smearing, or prolonged incubation at acid pH to demonstrate histochemical activity with lead salts.

Since latency is not present when azo dye methods are used, correlated light microscopy may be essential in some cases

(41).

D. Interpretation of Results

There are many problems in interpreting phosphatase cyto- chemical activity. In most tissues there are many different enzymes that hydrolyze a wide variety of phosphatase substrates

(28, 30, 38, 39). As mentioned previously, biochemical assays when possible can provide essential evidence in confirming the identity of an enzyme (40). In addition, the careful use of selective inhibition in control cytochemical experiments can also provide valuable information concerning the identity of an enzyme in question (38). For example, a brief preincubation of a specimen in buffer at an acid pH will inhibit glucose-6- phosphatase activity (which is sensitive to acid), whereas the activity of an acid phosphatase is unaffected (38).

There are multiple aryl sulfatases as well as phosphatases (42 - 44). It is significant that the genetic deficiency of aryl sulfatase A produces a clinically different mucopoly- saccharidosis from that produced by the genetic deficiency of aryl sulfatase B (43). As yet, however, which of the enzymes are demonstrated cytochemically by the Goldfischer method (42) remains uncertain (44).

IV. CONCLUDING REMARKS

Although cytochemistry for electron microscopy is a de- manding technique, it is rewarding in that it provides informa- tion that cannot otherwise be obtained. These methods are of greatest value when used to distinguish among organelles or cell populations that could not otherwise be distinguished

(i.e., by either biochemical or morphological techniques). For

example, cytochemistry has been used successfully to identify

functionally different cells in a mixed population (45, 46) and

to distinguish between morphologically similar organelles with

different functions (3, 47, 48). Although the number of methods

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V. MORPHOLOGY OF MONONUCLEAR PHAGOCYTES 425 available for cytochemistry remains limited, there are nonetheless many significant problems that can profitably be explored using cytochemistry for electron microscopy.

V. APPENDIX

A. Incubation Media

(1) Barka - Anderson Acid Phosphatase Incubation Medium (49) (Modified Gomori Method)

Stock solutions

1.25% 3-Glycerophosphate, Grade I (make fresh)

(Add 125 mg ß-glycerophosphate to 10 ml distilled H2O) Adjust to pH 5.0 with 1 N HC1

0.2 M Tris-maleate buffer, pH 5.0 (make fresh) 0.2% Lead nitrate (store at room temperature) Medium

(a) 10 ml Tris-maleate buffer (b) 10 ml distilled Water

(c) 10 ml Substrate (3-glycerophosphate)

(d) 20 ml Lead nitrate, added dropwise with constant stirring

Combine ingredients in the order listed above (a - d ) . Adjust pH to 5.0. Add 5% sucrose.

Controls

Without substrate: Make up medium as above but substitute additional 10 ml buffer for the substrate solution.

Sodium fluoride inhibition: Add 0.01 M NaF to the complete incubation medium.

(2) Aryl Sulfatase Incubation Medium (42)

Stock solutions

Acetate Veronal buffer, see Buffers, Section V.D.2.

24% Lead nitrate (store at room temperature) Medium

(a) 25 mg p-Nitrocatechol sulfate

(b) 5.0 ml Acetate Veronal buffer, pH 5.4

(c) 0.16 ml 24% Lead nitrate. Add slowly with stirring.

(Lead nitrate causes a precipitate, but it will go into solution at pH 5.7.) Adjust to pH 5.5 with 0.1 N HC1.

Add 5% sucrose.

Control without substrate: Make up medium as above but omit p-nitrocatechol sulfate. At present, a specific inhibitor of aryl sulfatase is not available.

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(3) Peroxidase Incubation Medium (50)

Medium

(a) 10 ml 0.05 M Tris-HCl buffer, pH 7.6

(b) 5 mg 3,3'-Diaminobenzidine tetrahydrochloride (c) 0.1 ml Diluted hydrogen peroxide (dilute 0.3 ml 30%

hydrogen peroxide to 10 m l ) . Add to medium just before use.

Adjust pH to 7.6. Add 5% sucrose. All solutions used in the incubation medium should be made the day that they are used.

Controls

Without substrate: Prepare incubation medium without hydrogen peroxide.

Inhibitors: The following inhibitors (0.01 M concentration) may be useful in the interpretation of peroxidase cytochemistry: potassium cyanide, sodium azide, and amino-triazole. One hour preincubation of fixed tissues with inhibitors in media lacking substrate may be neces- sary for penetration of the inhibitors into organelles such as leukocyte granules. Then incubate as usual in the complete medium with added inhibitor.

B. Fixatives

(1) 1.5% Glutaraldehyde in 0.1 /i sodium cacodylate-HCl, pH 7.4 + 1% s u c r o s e :

30 ml 10% Glutaraldehyde (E. M. Sciences) 100 ml 0.2 M Sodium cacodylate stock

70 ml distilled H2O. Total solution 200 ml. pH

to 7.4. Add 2 gm sucrose. Keeps 2-4 weeks refrigerated.

(2) Dilute Formaldenyde-glutaraldehyde ( 7 ) : 0.5 gm paraformaldehyde powder

19 ml distilled water

25 ml 0.2 M Sodium cacodylate stock

6 ml 2 5 % Glutaraldehyde. Total solution 50 m l . Warm paraformaldehyde and water for 30 min in a 70°C water bath with stirring. Add 4-5 drops 0.1 N NaOH. Swirl until clear and cool. Add cacodylate and glutaraldehyde. pH to 7.4. Add 25 mg CaCl2 just before u s e . Fix tissues either cold or at room temperature, usually 1-4 hr for optimal morphology. Fix only 5 min before cytochemical experiments.

(3) Palade's Osmium:

Mix just before use

4 ml Acetate-Veronal stock 4 ml 0.1 M HC1

2 ml distilled water. Total volume 10 m l . pH to 7.6.

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V. MORPHOLOGY OF MONONUCLEAR PHAGOCYTES 427 Add 10 ml osmium, 2% stock solution. Add 5% sucrose.

In Block Stain

Kellenberger's in Block Uranyl Acetate Stain (36) 20 ml Acetate-Veronal stock

28 ml 0.1 if HC1

52 ml distilled H2O. Total solution 100 ml.

Adjust pH to 6.0. Add 0.5 gm uranyl acetate to 100 ml of buffer. Store in refrigerator. Keep from light.

Add 4% sucrose to a small aliquot on day of use.

D. Buffers

(1) Acetate-Veronal (A-V) Stock

1.943 gm Sodium a c e t a t e (MW 136, formula c o n c e n t r a - t i o n 0.14 M)

2.943 gm Sodium Veronal (MW 206.18, formula concentration 0.14 M)

Dissolve and dilute to 100 ml with distilled H2Ö.

(2) Acetate-Veronal Buffer 20 ml A-V stock

20 ml 0.1 N HC1

Dilute to 100 ml with distilled H2O. Adjust to pH 5.4 for aryl sulfatase incubation medium.

(3) Michaelis Wash Buffer 20 ml A-V stock

20 ml 0.1 N HC1

Mix and dilute to 100 ml with distilled H2O. Adjust pH to 7.4. Add 7 gm reagent grade sucrose.

(4) 0.2 ii Sodium cacodylate Stock 21.4 gm sodium cacodylate

Add distilled H20 to make 500 ml.

(5) Sodium cacodylate-HCl Wash Buffer 50 ml 0.2 M Sodium c a c o d y l a t e stock

Dilute with distilled Η20 to 100 ml. Adjust pH to 7.4 with 0.1 N HC1. Add 7 gm sucrose.

Addresses of Chemical Companies J. T. Baker Chemical Company Phillipsburg, New Jersey 08865

(201) 859-2151

or: VWR Scientific, Inc.

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Electron Microscopy Sciences Box 251

Fort Washington, Pennsylvania 19034 (215) 646-1566

F i s h e r S c i e n t i f i c Co.

711 Forbes Ave.

Pittsburgh, Pennsylvania 15219 (412) 562-8300

Ladd Research Industries P.O. Box 901

Burlington, Vermont 05401 (802) 658-4961

Mallinckrodt, Inc.

P.O. Box 5439

St. Louis, Missouri 63147 (314) 231-8980

or: Scientific Products Sigma Chemical Co.

P.O. Box 14508

St. Louis, Missouri 63178

(800) 325-3010 (Order toll-free) Worthington Biochemical Corporation Freehold, New Jersey 07728

(800) 631-2142 (Order toll-free)

REFERENCES

1. M. A. Hayat (ed.). "Electron Microscopy of Enzymes.

Principles and Methods," Vols. 1-4. Van Nostrand Reinhold, New York, 1973-1975.

2. B. A. Nichols, D. F. Bainton, and M. G. Farquhar. Differ- entiation of monocytes. Origin, nature, and fate of their azurophil granules. J. Cell Biol. 50: 498-515, 1971.

3. B. A. Nichols and D. F. Bainton. Differentiation of human monocytes in bone marrow and blood. Sequential formation of two granule populations. Lab. Invest. 29: 27-40, 1973.

4. B. A. Nichols. Normal rabbit alveolar macrophages. II.

Their primary and secondary lysosomes as revealed by elec-

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V. MORPHOLOGY OF MONONUCLEAR PHAGOCYTES 429 tron microscopy and cytochemistry. J. Exp. Med. 144:

920-932, 1976.

5. D. F. Bainton, B. A. Nichols, and M. G. Farquhar. Primary lysosomes of blood leukocytes· In "Lysosomes in Biology and Pathology," Vol. 5 (J. T. Dingle and R. T. Dean, eds.), pp. 3-32. North-Holland, Amsterdam, 1976.

6. D. D. Sabatini, K. Bensch, and R. J. Barrnett. Cyto- chemistry and electron microscopy. The preservation of cellular ultrastructure and enzymatic activity by aldehyde fixation. J. Cell Biol. 17: 19-58, 1963.

7. M. J. Karnovsky. A formaldehyde-glutaraldehyde fixative of high osmolality for use in electron microscopy. J.

Cell Biol. 27: 137A-138A, 1965.

8. H. D. Fahimi and P. Drochmans. Essais de standarisation de la fixation au glutaraldehyde. I. Purification et détermination de la concentration du glutaraldehyde. J.

Microsc. 4: 725-736, 1965.

9. E. A. Robertson and R. L. Schultz. The impurities in commercial glutaraldehyde and their effect on the fixation of brain. J. Ultrastr. Res. 30: 275-287, 1970.

10. R. E. Smith and M. G. Farquhar. Lysosome function in the regulation of the secretory process in cells of the an- terior pituitary gland. J. Cell Biol. 31: 319-347, 1966.

11. D. Janigan. The effects of aldehyde fixation on acid phosphatase activity in tissue blocks. J. Histochem.

Cytochem. 13: 476-483, 1965.

12. P. M. Seeman and G. E. Palade. Acid phosphatase localization in rabbit eosinophils. J. Cell Biol. 34: 745-756, 1967.

13. A. Leskes, P. Siekevitz, and G. E. Palade. Differentiation of endoplasmic reticulum in hepatocytes. 1. Glucose-6-

phosphatase distribution in situ. J. Cell Biol. 49: 264- 287, 1971.

14. L. S. Kaplow. Buffy coat preparatory tube. Am. J. Clin.

Pathol. 51: 806-807, 1969.

15. R. van Furth (ed.). "Mononuclear Phagocytes, Functional Aspects. Parts I and II." Martinus Nijhoff Publishers, The Hague, 1980.

16. R. M. Steinman and Z. A. Cohn. The interaction of soluble horseradish peroxidase with mouse peritoneal macrophages in vitro. J. Cell Biol. 55: 186-204, 1972.

17. F. Roels, E. Wisse, B. De Prest, and J. van der Meulen.

Cytochemical discrimination between catalases and peroxidases using diaminobenzidine. Histochemistry 41: 281-312, 1975.

18. J. Breton-Gorius and J. Guichard. Améliorations techniques permettant de révéler la peroxydase plaquettaire. Nouv.

Rev. Fr. Hematol. 16: 381-390, 1976.

19. A. R. Spurr. A low-viscosity epoxy resin embedding medium

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