Lectin Methods and Protocols

563 Pages·2010·42.635 MB·English

by  Jonathan M. Rhodes, Jeremy D. Milton

1
Light Microscopy
Overview and Basic Methods
Anthony J. Leathem and Susan A. Brooks
1. Introduction
1.1. Why Use Lectins?
Lectms are proteins or glycoprotems of nonimmune origin derived from
plants, animals, or mlcroorgamsms that have specn7cIty for termmal or subter-
minal carbohydrate residues. They are sensitive, stable, and easy-to-use tools.
Lectm hlstochemlstry and cytochemistry can provide an extraordmarlly sense-
ttve detection system for changes m glycosylatlon and carbohydrate expres-
sion that may occur during embryogenesis, growth, and disease. Although
carbohydrates occur in a vast range of permutations that may be present m and
between cells, there 1s frequently a dominance and conservation of structures
to give specific markers of cell types or differentiation. Lectm hlstochemlstry
or cytochemistry can reveal subtle alterations in glycosylatlon between other-
wise mdistmgulshable cells.
Lectins wtll specifically recogmze and bmd to carbohydrate structures on
the surface of cells, on cytoplasmic and nuclear structures, and m extracellular
matrix m cells and tissues from throughout the ammal and plant kingdoms,
down to bacteria and vu-uses.
7.2. What Do Lectins Bind to?
Sugar combmmg specificity of lectins is usually quoted in terms of the
monosaccharide that best inhibits its binding to cells; for example, Helix
pomatia lectin is said to be specific for N-acetyl-galactosamine, concanavalm
From Methods m Molecular Medune, Vol 9 Lecbn Methods and Protocols
Edited by J M Rhodes and J D Melton Humana Press Inc , Totowa. NJ
3
4
A for mannose, and so on However, thts IS a massive overstmphficatton, and
the exact, complex structures recogmzed by most lectms remam unknown.
Lectms bmd approx 10 times more avidly to solid-phase or tmmobthzed sugars
as to the same sugars m solutton, which probably reflects the association-dts-
soctatton equtlibrmm between the lectin bmdmg sites and the sugar, and the
multtvalency of lectms A typical lectm binding site may consist of three
monosacchartdes, and mcludes then lmkage and adjacent protein or lipid back-
bone. Furthermore, a lectm will bmd mcreasmgly m lo-fold steps between
mono-, dt-, and trtsacchartdes.
1.3. Cell and Tissue Samples
Lectm htstochemtstry to detect alterations in cellular glycosylation can be
performed on hvmg cells m suspension, on cell smears, tissue tmprmts, or fresh
cryostat sections. Here, sugars m the extracellular matrix, glycohptds. and gly-
coprotems can be detected readily If archival tissues are of interest (e.g., in
which cellular glycosylatton 1s to be correlated with disease progressron or
prognoses m a retrospective study), then sections from formalm-fixed, paraf-
fin-embedded ttssues may be preferred Here, glycohpids will have been lost
during tissue processing and glycans may be sequestered during fixation and
processmg. For thts reason, glycans-although stall present-may be hidden
and unavailable for lectin bmdmg. Such sequestration can usually be at least
partially overcome by pretreatment with a dtgesttve enzyme such as trypsm,
which reveals hidden structures
1.4. The Range of Lectins Available
About 100 drfferent lectms are readily available commerctally, and hun-
dreds of others have been detected and described, mostly by their agglutmatton
of red cells. Many lectins have not been assessedf or then histochemtcal stam-
mg of tissues and cells.
In the United Kingdom, two suppliers with a wide range of lectms are Sigma,
Poole, Dorset, UK, and EY Laboratories (UK agents are Bradsure Btologtcals,
Market Harborough, Lercestershire).
There undoubtedly exist hundreds, thousands, or tens of thousands of lectms
in nature that remam to be discovered and purtfied. It IS very simple to produce
a crude lectm preparation m the laboratory that will give good results m
tmmunohistochemistry Excellent sources include virtually any beans or seeds,
and most green plant tissue like stems and leaves. The easiest method of
detecting lectin binding with such preparations, or with unlabeled commer-
cially purchased lectms m which no antibody 1s available, is through bto-
tinylation of the lectm
Light Microscopy 5
A
label (eg. FITC 01
carbohydrate
Fig. 1. The direct method.
1.5. Histochemical Methods
1.5.1. The Direct Method
The simplest methods for lectin histochemistry are the so-called direct
methods, which rely on detection of lectin binding through the presence of a
label conjugated directly onto the lectin molecule. The most commonly used
labels are either fluorescent labels such as fluorescein isothiocyanate (FITC),
which are visible when viewed under UV light, or enzyme labels like horserad-
ish peroxidase, which yield a visible, colored reaction product (see Note 1).
The principle of the direct method is illustrated in Fig. 1. The advantages of a
direct method is that it is quick, easy, and cheap. The disadvantages are that it
is generally less sensitive than the more complex methods (see Section 3.6.4.)
and that the incorporation of a relatively large labeling molecule, like horse-
radish peroxidase, may interfere sterically with the combining site of the lectin
and therefore slightly alter binding characteristics.
1.5.2. The Indirect Antibody Method
Here, the binding of native, unconjugated lectin is revealed by a second step
in which a labeled (again, fluorescent or enzyme label) antibody (usually
polyclonal, raised in rabbit) is added. The principle of this technique is illus-
trated in Fig. 2. The addition of a second layer has the advantage of increasing
sensitivity and removes the potential problem of the label molecule interfering
with binding specificity. Disadvantages are that the method takes longer, it is
slightly more expensive because extra reagents need to be purchased, and there
is slightly more potential for nonspecific background staining.
I. 5.3. Indirect Sandwich Methods
Further layers can be added. For example, native, unconjugated lectin can
be layered with first an unlabeled primary antibody (for example, a polyclonal
Leathem and Brooks
label (eg. FKC or peroxidase)
A
-rabbit anti-lectin antibody
Fig. 2. The indirect antibody method.
label (eg. FITC or peroxidase)
- rabbit anti-lectin ambody
lectm
arbohydrate
Fig. 3. An indirect sandwich method.
antibody raised in rabbit against the lectin), then a labeled second antibody (for
example, a peroxidase labeled polyclonal antibody raised in swine against rab-
bit immunoglobulins). The principles of this method are illustrated in Fig. 3.
Again, the advantage of adding extra layers is increased sensitivity. The disad-
vantages are, as before, that the method takes longer, is slightly more expen-
sive, and there is slightly more potential for nonspecific background staining.
Light Microscopy 7
-f!- label (eg. FITC or peroxidase)
avidin
biotin
lectin
carbohydrate
Fig. 4. The simple avidin-biotin method.
1.5.4. Simple Avidin-Biotin Method
Avidin-biotin techniques rely on the great avidity with which avidin (a pro-
tein originally derived form egg white) binds to biotin (one of the B group of
vitamins). Lectins can be purchased ready labeled with biotin, or can be
biotinylated in the laboratory using a simple method (see Sections 2.9. and
. . . .
3.5.). Streptavidin, derived from Stveptomyces avlduw, has largely replaced
avidin, as it gives a cleaner staining result.
The simplest avidin-biotin technique relies on detection of the biotinylated
lectin by streptavidin linked to a label such as peroxidase. The principle of this
technique is illustrated in Fig. 4.
1.5.5. Multis tep A vidin-Biotin Methods
The avidin-biotin interactions can be exploited in more complex multistep
techniques. For example, unlabeled lectin is layered with a biotinylated anti-
body directed against it (e.g., polyclonal antisera raised in rabbit), followed by
streptavidin peroxidase. Alternatively, lectin is layered first with unlabeled
primary antibody (e.g., polyclonal antisera raised in rabbit) and then a second
antibody (e.g., a polyclonal antibody raised in swine to rabbit immunoglobu-
lins) labeled with biotin. This, again, would be followed by streptavidin per-
oxidase. The principle of these techniques is illustrated in Fig. 5.
1.5.6. Other Methods
Variations on these techniques can be developed to suit individual needs. It
is probably better to start with a simple, quick, direct method before going on
to more complex systems. Companies such as Dako (High Wycombe, United
Leathem and Brooks
label (eg. FITC or peroxidase) label (eg. FITC or
peroxidase)
\
avidin -&
wine antl-rabbit IgG
biotin
rabbit anti-lectin
rabbit anti-lectin
antibody
Fig. 5. Examples of multistep avidin-biotin methods.
Kingdom) supply easy-to-use kits of the main steps in some of the more com-
plicated techniques, as well as a comprehensive range of antibodies.
1.6. A Philosophical Point Before Starting
Although numerous lectins have been commercially available for years,
remarkably little progress has resulted. Histologists seem reluctant to try some-
thing new and the majority of lectin publications report on the same IO or 12
lectins, frequently purchased as a lectin kit and chosen by the chemical com-
pany according to availability rather than a scientific basis. (So be daring!)
We recommend that interested researchers:
1. Start by using a well-characterized lectin that is almost certain to work.
2. Progress to try lesser known lectins.
3. Choose a biological phenomenon, a diagnostic problem, or some a priori
hypothesis to test.
4. Or, select one lectin and explore the staining of a range of tissues; or select one
tissue and explore a selection of lectins with a theme, such as galactose-binding
or fucose-binding lectins (see Note 2).
5. Look for new or little used and uncharacterized lectins.
1.7. Which Lectins to Use and Which to Avoid
To get started, choose a cheap, well-documented lectin that binds to a
wide variety of tissues. Concanavalin A is a splendid starter-it is cheap
Lght Microscopy 9
and bmds to most cells and tissues. Ulex europeus lectm UEAI is mterest-
mg rn that It bmds to blood vessel endothelmm. Peanut lectm is also a good
starter, as it binds very specifically to a variety of cell types, givmg very beau-
tiful stammg patterns.
Some lectms are hrghly toxtc, and should be avoided whenever possible.
Lectms from Ricuzus communzs,t he castor oil bean (also called Ricm), Abrus
precutonus, the Jaquerty bean (also called Abrm), and Vzscuma lbum mistle-
toe, are all extremely toxic
Toxtcity and hazard data are unavailable for most other lectms Generally,
although dust and aerosols should obviously be avoided, no spectal precau-
ttons are needed.
1.8. Which Tissues to Use
To get started, you need a readily avatlable, reproducible source of trssue
that binds a wade variety of lectms. Kidney is very good for this-even fresh
pig kidney from a supermarket. Epithehal rich tissue IS generally successful
Pituitary (for hormone precursors) and salivary glands and gut (different
mucms) usually give mterestmg staining patterns.
1.9. The Way Forward
Lectm histochemtstry 1sr eally a first step. Bmdmg of lectms to tissue sec-
tions can demonstrate differences or changes m glycosylatton, but then iden-
ttfication 1s more drfftcult By competittve mhtbttion of lectm bindmg by
mono- and ohgosacchartdes and by enzyme digestron of tissue sections before
lectm staining, it is possible to get some idea of the structures recognized; and
this may be extremely sensitive and clnucally useful But to identify the struc-
ture really needs extraction, separation, isolation of the carbohydrate, probably
linked to a protein or hpid, then expensive and time-consummg procedures
such as mass spectrometry, sequential enzyme digestion, and computer-assisted
comparison with other known structures
1.10. Examples of the Useful Application
of Lectin Histochemistry
1.10.7. Ulex europaeus Lectm UEAland Endothekd Cells
UEexe uropeus (gorse) has two main lectins-UEAI, which binds to fucose;
and UEAII, which bmds to N-acetyl glucosamme. UEAI 1sa n excellent marker
of human endotheltal cells m tissue sections and m culture. It IS probably a
better marker than the more commonly used Von Willebrand factor. UEAI
bmding gives intense stammg, particularly on fresh tissues or after trypsm
digest in paraffin sections. Not all endothebal cells stain-for example, those
10 Leathem and Brooks
m gut may be negative-but some, like those m capillartes and muscle, do so
mtensely The bmdmg IS not absolutely spectfic for endotheltal cells-for
example, normal intestinal mucosal cells may sometimes stam. Some cancers,
mcludmg prostate and pancreas, also stain strongly, presumably reflecting a
change m fucosylatton.
UEAI shows some species specifictty, and different lectms may be more
suitable for ammal endothelmm. Bandwea simplzczfolza isolectm 1 (BS-I),
peanut lectin, and wheat germ lectin all stain endothelmm m mouse, rat, pig,
and sheep, and work well-though not as well as UEAI-m human tissues too
I 10 2. Helix pomatia Lectin (HPA) and Cancer
The lectm from Helzxpomatza (the Roman or edtble snail) recognizes termi-
nal N-acetyl galactosamme, and to a lesser extent Wacetyl glucosamme and
galactose sugars. It appears to be a very good prognosttc marker m breast and
other cancers. Approximately 80% of breast cancers will stain strongly for the
bmdmg of the lectm, whereas approx 20% are completely negative The posi-
tive cancers tend to be aggressive, to spread to other sites (metastasize), and
have a very poor prognoses. The negative cases tend not to spread and have a
much more favorable prognosis (2,2). Studies have shown that HPA bmdmg is
also a marker of poor prognosis m other cancers, mcludmg colorectal (3,4),
gastric (.5), esophageal (6), and prostattc cancers (7). The HPA appears to rec-
ognize complex sugar(s) that are mvolved m aggresstve biologtcal behavior
and spread of cancers.
2. Materials
2.7. Lectin Buffer (see Note 3)
1. 60 57 g Tris (Sigma), 87 g NaCl, 2.03 g MgC12, 1 11 g CaCl, dissolved in 1 L
distilled H20.
2 Adjust pH to 7 6 using concentrated HCl
3 Make up to a total volume of 10 L with distilled water
2.2. Preparation of Tissue Imprints
1 Piece of fresh tissue approx 1 cm3 m size.
2 Glass microscope shdes
3. Acetone or methanol
2.3. Preparation of Cell Smears
1 Cells m suspension (e g., in ttssue culture fluid, any suitable buffer, or in body
flutds lake blood)
2 Glass microscope slides
3 Acetone or methanol
Light MIcroscopy 11
2.4. Preparation of Frozen (Cryostat) Sections
1 Piece of tissue approx 0 5 cm3 m stze
2 Glass microscope shdes
3 OCT embeddmg medium (Trssue-Tek, supphed by Raymond A. Lamb)
4 Isopentaneo r hexane
5, Liquid nitrogen
6 Acetone or methanol
2.5. Preparation of Forma/in-Fixed, Paraffin-Embedded Sections
1 Formalm-fixed. paraffin-embedded blocks of tissue
2 Glass microscope slides.
3 20% ethanol m drstrlled water.
4. CNP30 (BDH) or xylene
5 99% v/v and 70% v/v ethanol m dtsttlled water
6 Dtsttlled water.
7 Lectm buffer
2.6. Trypsinization of Paraffin Sections
Trypsm solution should be made fresh immediately before use Glassware,
buffers, and so on should be prewarmed to 3792, and trypsmtzation should be
carried out at 37°C either m a water bath or an incubator.
Dtssolve 400 mg trypsin (crude type II from porcine pancreas, Sigma) and
400 mg CaC12 m 400 mL lectm buffer warmed to 37°C. Filter. Immerse slides
m the solutron at once and incubate at 37°C m an incubator or water bath
(see Note 4).
2.7. Blocking Endogenous Peroxidase (see Note 5)
3% v/v solution of hydrogen peroxide m methanol. Make fresh as requtred.
2.8. Preparation of Crude Mushroom Lectin Extract
1, Mushrooms (the white, closed cup vartety stocked by most supermarkets work well).
2. Laboratory &sue homogenizer, or domestic food processor or Juicer
3 Lectm buffer or dtsttlled water.
2.9. Labeling with Biotin
1. O.lMNaHCOs m dlsttlled water
2 N-hydroxysuccnnmtdobtotin (NHS-biotm, Sigma) (see Note 6).
3 Dtmethylformamtde or dlmethyl sulfoxtde
4 Lectm buffer
2. IO. Siotinylated Mushroom Lectin Binding to Gut Sections
1 Paraffin or frozen sections of human or animal gut (materials listed m Section
2 4 or 2 5. and m 2.7 requrred)
12 Leathem and Brooks
2 Btotmylated crude mushroom extract (matertals listed m Sections 2 8 and 2 9
required)
3 Lectm buffer
4 Streptavtdm ConJugated with horseradrsh peroxtdase (Calbiochem, La Jolla, CA)
diluted l/400 m lectm buffer
5 3,3-diammobenzidme tetrahydrochlortde (DAB; Stgma). 0 5 mg/mL m lectin
buffer Hydrogen peroxide added to give a concentratton of 5% v/v immediately
before use (see Note 7)
6 Mayers hematoxylm solution (Sigma)
7 70% v/v, 95% v/v, and 99% v/v ethanol m distilled water
8 CNP30 (BDH) or xylene
9 Depex resinous mountmg medium (BDH)
2.11. Direct Method to Detect Peroxidase-Labeled
Peanut Lectin Binding to Kidney Sections
Paraffin or frozen secttons of human or animal kidney (materials listed m Section
2 4 or 2 5 required, materials listed m Sections 2 6 and 2 7 required)
Peroxidase-labeled peanut lectm (PNA. Sigma) at a concentration of 10 pg/mL
m lectm buffer
Lectm buffer
3,3-dtammobenztdme tetrahydrochlortde (DAB; Sigma) 0 5 mg/mL m lectm
buffer Hydrogen peroxide added to give a concentration of 5% v/v immediately
before use (see Note 7)
Mayers hematoxylm solution (Sigma).
70% v/v, 95% v/v, and 99% v/v ethanol in distilled water
CNP 30 (BDH) or xylene
Depex resinous mounting medium (BDH).
2.72. Direct Method to Detect FITC-Conjugafed
UEAI Binding to Endothelial Cells
1 Paraffin sections, frozen sections, or tmprmts of human striated muscle, or smears
of cultured human endothehal cells (materials listed m Sections 2 2.-2 4 , or 2 5.
required)
2 Fluorescem lsothtocyanate (FITC)-labeled Ulex europeus tsolectm I (UEAI)
(Sigma) at a concentration of 10 yg/mL m lectm buffer
3 Lectm buffer
2.13. Indirect Antibody Method
to Detect UEAI Binding to Endothelial Cells
1 Paraffin sections, frozen sections, or tmprmts of human striated muscle, or smears
of cultured human endothelial cells (materials hsted m Sections 2 2 -2.4 , or 2 5
reqmred; materials listed in Sections 2 6 and 2.7 required)
2 Ulex europeus tsolectm I (UEAI; Sigma) 10 pg/mL m lectm buffer