Lectins for histochemical demonstration of glycans

Abstract

Lectins have been proven to be invaluable reagents for the histochemical detection of glycans in cells and tissues by light and electron microscopy. This technical review deals with the conditions of tissue fixation and embedding for lectin labeling, as well as various markers and related labeling techniques. Furthermore, protocols for lectin labeling of sections from paraffin and resin-embedded tissues are detailed together with various controls to demonstrate the specificity of the labeling by lectins.

Appendices

Appendix 1: Chemical fixation, embedding and mounting of sections

Cell monolayers or cell suspensions are fixed in 2% paraformaldehyde–0.1% glutaraldehyde in PBS (10 mM phosphate buffer, pH 7.2, 0.15 M NaCl) or 0.1 M cacodylate buffer (pH 7.2) for 30 min at culture temperature. Formaldehyde freshly made from paraformaldehyde and vacuum-distilled glutaraldehyde should be used. Aliquots of the fixative can be stored at −20°C. The pH of the fixative needs to be checked before use.

For the detection of intracellular lectin-binding sites, cells must be permeabilized. The most useful permeabilization reagent is saponin. Depending on cell type, 0.1–0.3% saponin in buffer containing 0.1% bovine serum albumin is applied for 10 min. Since permeabilization by saponin is reversible, it is recommended to add saponin to the lectin and other incubation solutions. Triton X-100 is another useful detergent for permeabilization. Since it is a strong detergent, due care should be exercised to control the degree of extraction due to permeabilization.

Solid organs such as liver, kidney or brain should by fixed by vascular perfusion via the left cardiac ventricle. To remove blood, perfusion is started with Millipore-filtered (0.22 μm) and oxygenated PBS or cacodylate buffer containing 4% PVP (≥40 kD) and 70 mM NaNO₂ until the organ is blanched. Perfusion then continues with 3% paraformaldehyde–0.1% glutaraldehyde in PBS or cacodylate buffer containing 4% PVP and 70 mM NaNo₂. The fixative must be Millipore-filtered and oxygenated before use. Perfusion is performed with solutions warmed to 37°C and at a hydrostatic pressure of about 110 mmHg. Perfusion with the fixative should last for ≥15 min and continued by immersion fixation of thin tissue slices for ≥1 h at ambient temperature.

The mucosal linings of organs such as stomach, intestine, bladder etc. can be easily fixed by immersion. For this, 3% paraformaldehyde–0.1% glutaraldehyde in PBS or cacodylate buffer containing 4% PVP is used. Fixation should last for ≥1 h initially at 37°C.

In any case, fixation is followed by rinses with buffer and incubation of the cells or tissue pieces in a solution to quench free aldehyde groups. This quenching step serves a twofold purpose in which the fixation is terminated in a controlled manner and non-specific binding of lectins to free aldehyde groups is prevented. A useful quenching solution is a freshly made 50 mM NH₄Cl in PBS. Quenching is performed on ice for 30 min. Other quenching solutions are 0.1 M glycine or borohydrate.

Cell pellets or tissue pieces can be embedded in paraffin or Epoxy resin according to the standard protocols, at progressively lowered temperature in Lowicryl K4M resin (Carlemalm et al. 1982; Roth et al. 1981, 1989) or in London resins (Newman and Hobot 1987).

For mounting the sections of paraffin-embedded samples, protein-coated glass slides or Superfrost^® slides are useful. Semithin sections (0.1–1 μm) of Lowicryl K4M or LR White/Gold-embedded samples can be conveniently attached to poly-l-lysine (300–500 kD)-coated glass slides. For mounting, semithin sections are placed with a wire loop onto a water drop over a diamond pencil-marked region of the poly-l-lysine activated glass slide and allowed to dry overnight at 40°C. Ultrathin sections from Lowicryl K4M or LR White-embedded tissues are prepared using glass knifes and placed on Parlodion/carbon-coated nickel grids.

Appendix 2: Incubation protocols

Paraffin sections are deparaffinized and rehydrated according to the standard protocol. Sections are then conditioned with PBS containing 1% bovine serum albumin, 0.005% Triton X-100 and 0.05% Tween 20 for 10 min at ambient temperature.

Prior to lectin labeling of semithin Epon sections, the resin must be partially removed. Controlled resin removal can be achieved by using different alkaline alcohol solutions. Alcohol KOH solution is prepared by dissolving 2 g of KOH pellets in a mixture of 10 ml of absolute methanol and 5 ml of propylenoxide in a glass beaker using a magnetic stirrer. Similarly, alcohol NaOH solution is made by dissolving 2 g of NaOH pellets in a mixture of 10 ml of ethanol and 5 ml of propylenoxide. Before use, filter the alkaline alcohol solutions using ordinary filter paper. Following controlled, partial resin removal (about 2–5 min), semithin sections on glass slides are rinsed with 50% methanol or ethanol (to prevent detachment of sections), twice in water and then with PBS. Condition sections with PBS containing 1% bovine serum albumin and 0.05% Tween 20 for 10 min at ambient temperature.

Nota bene Alkaline alcohol solutions are not only irritant but also highly corrosive. Appropriate precautions should be taken to prevent contact with skin, wear eye protection. Also note that an exothermic reaction may occur during the preparation of the alkaline alcohol solutions. Alkaline alcohol solutions are highly volatile and should be used in a fume hood. Observe the sections on the slides during resin removal and add alkaline alcohol solution as required.

Semithin Lowicryl K4M sections do not require etching/resin removal prior to labeling. Sections are conditioned with PBS containing 1% bovine serum albumin and 0.05% Tween 20 for 10 min at ambient temperature.

Direct labeling with lectin–gold complexes

After draining the conditioning solution, sections are incubated with lectin–gold complexes for 45–60 min in a humidity chamber at ambient temperature. The optimal concentration of the lectin–gold complexes has to be empirically determined. For reproducible results, lectin–gold complex concentration is best expressed as OD_525 nm, which is the maximum absorption of colloidal gold. To start, test dilutions with an OD_525 nm between 0.05 and 0.1. The optimal dilution also depends on the gold particle size. As a rule of thumb, the larger the gold particles are the higher is the required OD. Wash sections in PBS (2 × 5 min each) and then immerse them in 1% glutaraldehyde for 30 min to prevent loss of lectin–gold complexes during silver amplification. Rinse the sections quickly with PBS and thoroughly in several changes of double-distilled water, and air-dry them. Air-dried sections can be stored as required or can be processed for silver amplification (see below).

Indirect labeling with lectin–digoxigenin conjugates

Drain conditioning buffer and avoid drying of sections. If horseradish–peroxidase conjugated anti-digoxigenin antibody is used, endogenous peroxidase activity must be blocked by treating sections with 0.3% H₂O₂ in methanol for 30 min at ambient temperature followed by rinses with PBS (2 × 5 min each). After conditioning, cover the sections with lectin–digoxigenin conjugate and incubate them for 45–60 min in a humidity chamber at ambient temperature. The optimal concentration of the lectin–digoxigenin conjugate depends on the lectin used (examples are given in Table 1). Wash sections in PBS (2 × 5 min each). Binding of the lectin–digoxigenin conjugate can be visualized with gold-labeled anti-digoxigenin antibody or with horseradish–peroxidase-conjugated anti-digoxigenin antibody.

Gold-labeled anti-digoxigenin antibody

Incubate sections with gold-labeled anti-digoxigenin antibody diluted in PBS containing 1% bovine serum albumin and 0.05% Tween 20 for 1 h at ambient temperature in a moist chamber. For 8-nm antibody–gold complexes, dilute to an OD_525 nm = 0.05; for 15-nm antibody–gold complexes, dilute to an OD_525 nm = 0.1. Afterward, rinse sections in PBS (2 × 5 min each) and immerse slides with sections in 1% glutaraldehyde for 30 min. Rinse sections quickly with PBS and then thoroughly with several changes of double-distilled water. Air-dry sections and process them for silver amplification.

Horseradish–peroxidase conjugated anti-digoxigenin antibody

Incubate tissue sections with appropriately diluted horseradish–peroxidase-conjugated antibody (e.g., 0.3 U/ml; (Sata et al. 1990b) for 45 min at ambient temperature. Afterward, rinse sections in PBS (2 × 5 min each) and reveal peroxidase activity with the diaminobenzidine reaction (see below), counterstain and mount sections.

Indirect labeling with glycoprotein-gold complexes

Some lectins, such as Concanavalin A, wheat germ lectin, Limax flavus lectin and Amaranthus caudatus lectin, can be conveniently used in an indirect labeling protocol, which applies glycoprotein–gold complexes for interaction with native lectins bound to tissue sections. After conditioning, the sections are covered with lectin diluted in PBS containing 1% bovine serum albumin and 0.05% Tween 20 and incubated for 45–60 min in a humidity chamber at ambient temperature. The optimal concentration of the lectin has to be empirically determined. We use 10 or 20 μg/ml Concanvalin A or wheat germ lectin, ≤100 μg/ml Datura stramonium lectin, ≤100 μg/ml Limax flavus lectin, and 10 μg/ml of Amaranthus caudatus lectin. Afterward, sections are rinsed in PBS (2 × 5 min each) and incubated with glycoprotein–gold complexes diluted with PBS containing 1% bovine serum albumin and 0.05% Tween 20 to OD_525 nm between 0.1 and 0.2. Horseradish–peroxidase–gold complex is used with Concanvalin A, ovomucoid–gold complex with wheat germ agglutinin, fetuin–gold complex with Limax flavus lectin and asialoglycophorin– or asialomucin (from bovine submandibular gland)–gold complex with Amaranthus caudatus lectin. Following rinses in PBS (2 × 5 min each), slides with sections are immersed in 1% glutaraldehyde for 30 min, rinsed quickly with PBS and thoroughly in several changes of double-distilled water. Following air-drying, silver amplification is performed (see below) and sections are counterstained as required, for instance with nuclear fast red, and mounted.

Silver amplification

Kits for the silver amplification of colloidal gold labeling can be obtained from various commercial sources. A relatively light-insensitive and easy-to-perform laboratory procedure with the use of silver acetate is as follows:

Solution A make fresh 0.2% w/v silver acetate (Fluka) in double-distilled water. Dissolve using a magnetic stirrer in an Erlenmeyer flask covered with aluminum foil. Note Silver acetate dissolves slowly; hence prepare at least 30 min in advance.

Solution B make 0.5% w/v hydroquinone in citrate buffer (0.05 M, pH 3.8). Generally make twice as much solution B as solution A.

Place slides with the attached tissue sections for 2–5 min in solution B to which an equal volume of double-distilled water has been added. Then transfer slides into a solution consisting of equal volumes of solution A and B and incubate for 18–22 min at 20–22°C. Afterward, rinse briefly with double-distilled water and place slides in diluted photographical fixative (from Agfa or Ilford) for 2–3 min. Finally, rinse thoroughly with tap water and counterstain the sections as required, and mount.

Diaminobenzidine reaction and heavy metal intensification of DAB reaction

To reveal catalytic activity of peroxidase, numerous oxidizable substances can be used, with 3,3′-diaminobenzidine tetrahydrochloride (DAB) being the most widely applied one. The conventional DAB reaction results in a brownish reaction product, whereas its intensification by heavy metals produces a dark brown/black reaction product.

For the DAB reaction, slides with the attached sections are transferred to 0.05 M Tris–HCl buffer (pH 7.6) for 10–30 min. Then, they are incubated in the dark in 0.5 mg/ml of DAB in 0.05 M Tris–HCl buffer for 10 min, which is followed by the full reagent composed of 0.5 mg/ml DAB and 20 μl 1% H₂O₂ in 0.05% Tris–HCl buffer for 30 min. After rinses with distilled water (3 × 5 min each), the sections can be counterstained as required and mounted.

For the heavy metal intensification of the DAB reaction (Adams 1981), dissolve 100 mg DAB in 200 ml 0.1 M phosphate buffer (pH 7.3) under stirring and add 5.0 ml 1% cobalt chloride slowly and drop-wise and then 4 ml 1% nickel ammonium sulfate. Place slides in this DAB-heavy metal mixture for 15–20 min. Afterward, add 0.66 ml 3% H₂O₂ and continue the incubation for additional 10–15 min. Finally, rinse the sections with 0.1 M phosphate buffer, counterstain as required and mount.

Ultrathin Lowicryl K4M or LR sections

Ultrathin sections are rehydrated and conditioned by placing the grids on droplets of PBS containing 1% bovine serum albumin and 0.05% Tween 20 for 10 min at ambient temperature. No etching/resin removal prior to lectin labeling is required. The incubation conditions for ultrathin Lowicryl K4M sections are identical with those described above for semithin Lowicryl K4M sections, except that no silver amplification is required. After completing the incubation, the grids are jet-washed with PBS (2 × 5 min each) and distilled water and let to air dry at ambient temperature overnight. Thin sections are counterstained with 3% aqueous uranyl acetate (4–5 min) and Millonig’s lead acetate (about 45 s) as detailed elsewhere (Roth 1989).

Appendix 3: Controls for specificity of lectin labeling

Pre-incubation of lectins with carbohydrate inhibitors is a classical protocol. For this, lectins are incubated for 30–60 min with mono- or disaccharides, synthetic oligosaccharides and neoglycoproteins or purified glycoproteins at concentrations ranging between 0.1 and 100 mM. Greatly reduced or absent labeling was observed with 5 mM methyl-α-mannopyranoside for Concanavalin A, 20 mM N-acetylglucosamine, N,N′-diacetylchitobiose or N-acetyllactosamine for Datura stramonium lectin, 50 mM d-galactose for peanut lectin, 10 mM N-acetylgalactosamine for Helix pomatia or soybean lectin, 10 mM N-acetylneuraminic acid for Limax flavus lectin, 0.1 mM 6′sialyllactose for Sambucus nigra and Polyporus squamosus lectin, 5 mM 3′sialyllactose for Maackia amurensis lectin, and 10 mM synthetic T antigen or T-antigen neoglycoprotein for Amaranthus caudatus lectin.

Deglycosylation by glycosidase pretreatment of sections. It is important that the glycosidases are protease-free. The various exoglycosidases should be applied as recommended by the manufacturer. For instance, treat sections with V. cholerae sialidase (0.02 U/ml in 50 mM acetate buffer, pH 5.5) for 18 h at 37°C in a moist chamber, rinse with distilled water followed by conditioning buffer. Run a control in enzyme-free buffer under identical conditions.

For the hydrolysis of the core-linkage between asparagine and N-acetylglucosamine of N-glycans, the sections are treated with 0.1% SDS in Tris-buffered saline at 70°C for 30 min and then washed in NP40 in Tris-buffered saline and incubated with PNGase F (80 U/ml in 0.1 M phosphate buffer, pH 7.4) for 18 h at 37°C in a moist chamber. Afterward, the sections are rinsed with PBS and conditioning buffer.

For the removal of high mannose-type N-glycans, sections are treated with 0.5 M citrate buffer (pH 5.5) containing 1% bovine serum albumin, followed by a rinse with citrate buffer and incubation with endo H (1 mU in citrate buffer) for 24 h at 37°C in a moist chamber. Afterward, the sections are rinsed with citrate buffer (3 × 5 min each), distilled water (3 × 1 min each) and conditioning buffer.

For the sequential enzymatic degradation of O-glycans, sections are treated with cacodylate buffer (10 mM, pH 6.0) and incubated with a glycosidase mixture in cacodylate buffer for 24 h at 37°C in a moist chamber. The glycosidase mixture consists of sialidase (0.03 units/ml), fucosidase (0.02 units/ml), α-N-acetylgalactosaminidase (0.25 units/ml), and endo-α-N-acetylgalactosaminidase. Afterward, the sections are washed with cacodylate buffer (2 × 5 min each) and distilled water.

Galactose oxidase pretreatment can be used to interfere with the lectin binding to terminal galactose residues. Sections are treated with 50 U/ml of galactosidase oxidase in PBS for 4 h at 37°C or with 5 U/ml overnight at 37°C. This can be combined with Schiff reagent for 15 min at ambient temperature.

Acid hydrolysis pretreatment to remove sialic acids. For this, sections are treated with 0.1 N H₂SO₄ for 1 h at 80°C. Afterward, they are rinsed with distilled water (5 × 5 min each) and covered with conditioning buffer and incubated with lectin.

Alkaline cleavage of the core-linkage between serine/threonine and O-glycan by the β-elimination reaction. Sections are treated with 0.1 N or 0.2 N NaOH for 12–36 h at 37°C in a moist chamber. Afterward, they are rinsed with distilled water (4 × 5 min each) and cover with conditioning buffer. This treatment removes O-glycans.

Saponification of sections to de-acetylate sialic acids. Sections are treated either with 0.5% KOH in 70% ethanol or with 0.1 N NaOH for 20 min at ambient temperature. Afterward, they are rinsed with distilled water (4 × 5 min each), covered with conditioning buffer and incubated with lectin.