Plasma cells and intracellular immunoglobulin

8.4 Plasma cells and intracellular immunoglobulin

Preparation in advance Prime mice 7–8 days before the experiment with 400 µg of alum-precipitated DNP–FγG (dinitro-

phenyl–fowl γ-globulin) plus Bordetella pertussis.

8.4.1 Demonstration of plasma cells

This can be done either on spleen sections or with single-cell suspensions smeared on microscope slides.

Frozen sections

MATERIALS AND EQUIPMENT Immune mouse (immunized, see Section 11.2.2) Acetone Solid carbon dioxide (dry ice) OCT (optimal cutting temperature) compound, Tissue Tek II Freezing microtome

METHOD

1 Add small pieces of solid CO 2 to acetone in an insulated metal container until bubbling stops.

2 Kill the mouse, remove the spleen and chop it transversely into about five pieces.

3 Drop the spleen fragments into the freezing mixture.

4 Mount one of the fragments on a microtome chuck using OTC compound and cut 5-µm sections. These must be air-dried and can then be stored at –70°C for up to

3 months.

Isolated cells

MATERIALS AND EQUIPMENT Immune mouse Tissue culture medium at 4°C Fetal bovine serum (FBS) Cytocentrifuge

8.4PLASMA CELLS AND INTRACELLULAR IMMUNOGLOBULIN

METHOD

1 Kill the mouse, remove the spleen and prepare a single-cell suspension in tissue culture medium.

2 Allow the aggregates to settle for 10 min at 1 g. Do not filter the cells through nylon wool because you are going to look at large cells which are easily damaged and trapped in nylon wool.

3 Wash the cells twice with medium by centrifugation (150 g for 10 min at 4°C).

4 Count the cells and adjust to 2 × 10 7 /ml.

5 Add an equal volume of FBS to the cell suspension.

6 Prepare cell smears on a cytocentrifuge. Spin at 100–150 g for 20 min. It will be necessary to vary the number of drops of cell suspension used to obtain a good smear; however, the total volume of liquid should be maintained at 4 drops per well (see also Section 11.6.3).

7 Dry the smears thoroughly and fix in 95% methanol. It is possible to prepare these smears as described for small lymphocytes, but the plasma cells are large and fragile, so you must not use a ‘spreader’ slide. Simply shake the slide to spread out the drop and air dry before methanol fixation.

8.4.2 Staining of sections or cytosmears for antibody-producing cells

Here it is demonstrated that there are antibody-producing cells present, and many of these cells are specific for antigenic determinants on the immunizing antigen, either carrier (FγG) or hapten (DNP).

MATERIALS Frozen section or smears from immunized mice Fluorescein isothiocyanate (FITC)-conjugated rabbit anti-mouse immunoglobulin Guinea-pig or pig-liver powder Phosphate-buffered saline (PBS)

There is a serious problem of non-specific adsorption of conjugated antisera to fixed material, in contrast to the very low background associated with viable cell immunofluorescence. You should use antisera with a low substitution ratio and in addition absorb the antiserum with liver powder as follows.

METHOD

1 Dilute the antiserum 1 : 5 with PBS and absorb 1 ml with 100 mg of pig-liver powder (use pro rata).

2 Mix for 30 min at 4°C and spin off the liver powder (500 g for 20 min at 4°C). This absorption must be done at the beginning of each staining session.

8.4.3 Detection of intracellular immunoglobulin

1 Dilute 2 aliquots of the absorbed antiserum to a final dilution of 1 : 10, 1 : 20 and 1 : 40 with PBS.

C H A P T E R 8: Lymphocyte structure

2 Apply 1 drop of each FITC conjugate dilution to separate spleen sections or cytosmears.

3 Incubate for 20 min at room temperature.

4 Wash off the excess antiserum. This can be done by putting the slides in a tray (face up!) and flooding them with PBS. Washing can be made more effective by placing the tray over a mag- netic stirring platform with the mixing bar at the extreme end of the tray from the slides. Mix slowly for 5 min.

5 Change the PBS and mix for a further 5 min.

6 Add 1 drop of mounting medium and add a coverslip. Ring with nail varnish.

7 Examine the slides under a UV microscope. Plasma cells should be easily visualized as shown in Fig. 4.2.

TECHNICAL NOTES • You may see a high background reaction when staining spleen sections for immunoglobulin

because of secreted antibody entrapment. This should not be a problem with cytosmears. • Slides of tissue sections may be stored at –20°C for several weeks after drying. Remove from the deep freeze and dry before use. • The antigen detected above (immunoglobulin) is relatively insensitive to drying-induced denaturation. For long-term storage, cells may be fixed after smearing. • Sometimes antigen reactivity can be lost if cells are allowed to dry out before or after fixation

(Fig. 4.3a,b). In this case, apply a concentrated suspension of cells to a slide pretreated with 1% w/v solution of poly L -lysine (to aid adhesion), allow them to settle at room temperature in a humid chamber for 5–10 min and then add an aqueous fixative.

• An essentially similar technique can be used for staining paraffin-embedded tissue sections with monoclonal antibodies, thus gaining additional information from the better preservation of histological structure.

We have described a direct immunofluorescence technique as this usually gives sufficient sens- itivity to detect the relatively large amount of immunoglobulin in the average plasma cell.

An indirect technique may be used if the antigen is scarce or poorly immunogenic. The first antibody, in this case rabbit anti-mouse immunoglobulin, is unconjugated. Its binding is visual- ized by a second antibody, e.g. FITC-conjugated goat anti-rabbit immunoglobulin (as illustrated in Fig. 4.2). The indirect technique gives a significant gain in sensitivity (up to eight second antibodies may bind for each first antibody), with only a marginal increase in the background of non-specific fluorescence. It also has the added convenience of having to prepare only a single conjugate of an antiserum from a large animal rather than a series of direct conjugates. For example, a FITC-conjugated IgG fraction of goat or sheep anti-rabbit immunoglobulin may be used to visualize a range of rabbit antisera to different antigens.

However, the technique is time-consuming and can be cumbersome if used for the simultane- ous detection of different antigenic determinants in two-colour immunofluorescence. If using indirect immunofluorescence to stain tissue sections, only the fluorescent conjugate need be absorbed with liver powder.

8.4.4 Detection of specific antibody using fluorescent probes

To demonstrate the antigen-binding specificity of intracellular antibody, incubate spleen sec- tions or cell preparations with fluorescein-conjugated FγG or unconjugated FγG visualized by

8.4PLASMA CELLS AND INTRACELLULAR IMMUNOGLOBULIN

FITC-conjugated rabbit anti-FγG antibody. We have described the indirect technique, which is more sensitive.

MATERIALS Frozen sections or smears from mice immunized with fowl g-globulin

Fowl g-globulin (FgG) Fluorescein-conjugated anti-FgG

METHOD

Again you should absorb the fluorescent conjugate with liver powder and, in addition, absorb or ultracentrifuge the FgG as this is also ‘sticky’.

1 Dilute FgG to 1 mg/ml and put 1 drop onto each of three sections or cytosmears of FgG- immune spleen.

2 Incubate for 20 min at room temperature.

3 Wash away the unbound antigen with PBS (see Section 8.4.3).

4 Add 1 drop of 1 : 5, 1 : 10 and 1 : 20 fluorescein-conjugated anti-FgG to each slide, respectively, and incubate for 20 min at room temperature.

5 Wash away the excess conjugate, blot dry and mount.

6 Examine the preparations under a UV microscope.

TECHNICAL NOTE It is important to ensure that the FITC-conjugated rabbit anti-FgG does not cross-react with mouse

immunoglobulin, i.e. it does not bind directly to the contents of the murine plasma cells. If it does, absorb with glutaraldehyde-insolubilized mouse g-globulin.

8.4.5 Detection of plasma cells with hapten–enzyme conjugates

This technique is similar in principle to that used for anticarrier antibodies (see Chapter 4). However, in this instance the detection molecule (enzyme) is much bigger than the antigen (the hapten, DNP).

Preparation in advance This method works equally well using horseradish peroxidase (HRP) or alkaline phosphatase (AP).

Conjugate the enzymes with DNP according to the method in Chapter 4. Although AP may be conjugated at room temperature (optimum substitution ratio DNP l0 –15 AP), HRP should be con- jugated at 4°C to slow the rate of reaction and so obtain the necessary low substitution ratio (optimum DNP 1–2 HRP). Enzyme activity is lost if HRP is oversubstituted, probably because of the addition of DNP groups into the catalytic site. The technique is designed to detect intracellular anti-DNP antibodies (see also Section 4.8.1).

TECHNICAL NOTE Determine the 280 and 360 nm absorbance values before conjugation as HRP has significant absorbance up to 403 nm. It is necessary to subtract the initial absorbance reading at 360 nm from that obtained after conjugation, to calculate the true degree of dinitrophenylation.

C H A P T E R 8: Lymphocyte structure

MATERIALS AND EQUIPMENT Cryostat sections (or cytosmears) from mouse immunized with DNP on a carrier protein Acetone Methanol Hydrogen peroxide Hapten–enzyme conjugate, e.g. dinitrophenyl–horseradish peroxidase (DNP–HRP) Diaminobenzidine (DAB) (Take care athis is a carcinogen) Phosphate-buffered saline (PBS) Bovine serum albumin (BSA)

METHOD

1 Air dry the cryostat sections and fix for 30 min in acetone containing 0.2% v/v hydrogen peroxide at room temperature to inactivate any endogenous peroxidase.

2 Rinse three times in PBS and wipe the slide around the section.

3 Overlay with DNP–HRP diluted to 0.5 mg/ml in PBS containing 0.1% w/v BSA. Leave for

30 min at room temperature in a humid atmosphere.

4 Wash in PBS and finally wipe the slide around the section.

5 Prepare the DAB substrate by adding 4 µl hydrogen peroxide to 10 ml PBS containing 6 mg DAB; filter and use immediately.

6 Add a few drops of DAB solution to each section and allow the colour reaction to develop for 20 min at room temperature in a humid chamber.

7 Wash carefully under tap water and counterstain, e.g. with Harris haematoxylin.

8 If permanent mounts are required, the sections may be dehydrated through graded alcohols, treated with xylene and mounted in DePeX.

9 Observe the sections under bright-field illumination; cell nuclei should be blue while plasma cells containing anti-DNP antibody should have blue cytoplasm.