Cell fixation and permeabilization

4.5 Cell fixation and permeabilization

A careful control of the procedures for cell fixation and permeabilization by removal of some or all of the membrane lipid is critical, particularly with monoclonal antisera. In the past a fixative might have destroyed the majority of epitopes recognized by a polyclonal antiserum; however, sufficient might remain to allow a visible antigen–antibody interaction still to take place.

For general use, fix cells with 4% paraformaldehyde in 0.1 m phosphate buffer, pH 7.4, for

20 min at room temperature followed by treatment with an organic solvent (methanol, acetone, etc.), as a way of optimizing structure preservation and permitting good permeabilization for antibody penetration for most mammalian cell types. The serological reactivity of surface H-2 immunoglobulin-receptor molecules and Thy-1 antigens is retained after fixation if the buffered paraformaldehyde solution is supplemented with paraperiodic acid (9 mg/ml) and 0.1 m l-lysine. Some antigenic determinants are destroyed by this procedure; in this case we recommend fixation directly in acetone for 5 min at −20°C.

Effective permeabilization can also be achieved by treating cells with a detergent solution (1% v/v Triton X-100 or Nonidet P-40) after formaldehyde fixation. Highly insoluble cellular pro- teins, such as those found in the cytoskeleton, may be prepared for immunofluorescent staining by detergent treatment without fixation (Fig. 4.3c).

It should be emphasized that time spent in optimizing fixation, permeabilization and staining techniques for each individual monoclonal antibody will usually yield dividends in the quality of the final result.

4.5CELL FIXATION AND PERMEABILIZATION

4.5.1 Permeabilization of cells, for example in preparation for flow

cytometry (adapted from Lan et al. 1996) To probe the contents of a cell, it is necessary to render the cell membrane permeable whilst still

endeavouring (as much as possible) to maintain the antigenicity and structure of the cell. A vari- ety of techniques are available and it is advisable for the researcher to identify the best method for the particular preparation of cells being investigated.

MATERIALS AND EQUIPMENT Cultured cells Phosphate-buffered saline (PBS) Absolute methanol Paraformaldehyde 0.5% v/v Nonidet P-40 (NP-40) in PBS 0.1% w/v saponin in PBS

0.01 M sodium citrate buffer, pH 6.0, containing 0.5% w/v bovine serum albumin (BSA) Microwave oven (800 W; 2450 MHz) 50-µm filter Polypropylene tubes

METHOD

1 Wash cells twice in PBS.

2 Resuspend the cells at a concentration of 10 6 cells/ml.

3 Fix the cells using either: (a) absolute methanol at –20°C for 15 min. Spin the cells at 1000 g for 5 min at room

temperature and remove the methanol. Add an equal volume of 0.5% NP-40 in PBS for 5 min to permeabilize cells then resuspend in PBS; or

(b) 2% paraformaldehyde on ice for 30 min. Spin cells at 1000 g for 5 min at room

temperature to remove the paraformaldehyde solution. Permeabilize the cells with 0.1% saponin in PBS for 5 min, then resuspend in PBS; or

(c) 2% paraformaldehyde on ice for 30 min. Remove the paraformaldehyde solution. Resuspend in PBS. Microwave the cells (see below).

Microwave treatment of cells

4 Suspend cells in 15 ml 0.01 M sodium citrate buffer, pH 6.0, containing 0.5% w/v BSA in unsealed 50-ml polypropylene tube.

5 Put the tube in a 1-litre pyrex beaker and cover with cling film wrapping, then place in the microwave oven.

6 Heat the cell suspension at maximum power (800 W) for 30–60 s.

7 Recover any straying cells from around the beaker and add to the cells that remained in the polypropylene tube.

8 Chill the cell suspension on ice for 10 min. Wash in PBS.

9 Filter through a 50-µm filter (this step helps to remove aggregated cell debris). Cells are now prepared for labelling with antibody.

C H A P T E R 4: Antibodies as probes

TECHNICAL NOTES • Advantages of microwaving cells include:

(i) Better preservation of cell morphology compared with other methods of permeabilization. (ii) Adequate permeabilization, thereby allowing large molecules into the cell for detection of

intracellular contents. (iii) Production of a distinct DNA profile for the analysis of DNA content. • Disadvantages of microwaving cells include: (i) Antigens are denatured by microwave treatment. (ii) Reduction of the microwave treatment time in order to limit the extent of denaturation may

mean a reduced level of detection of intracellular antigens.