Immunohistochemical detection of Bel2 protein in cultured cells

The overall procedure for immunostaining cultured cells is similar to that used for tissue sections in paraffin. The fixative solutions are more dilute and/or are used for only a short time, owing to the rapid penetration. Because most tissue culture medium contains biotin, which can cause non-specific staining when using avidin-biotin-based detection methods, an alkaline phosphatase detection system or Peroxidase Anti-Peroxidase Complex (PAP) (35) can be employed as an alternative to DAB. The sensitivity with these methods, however, is generally less than with DAB and therefore DAB should be tried first.

7.1 Procedures 7.1.1 Adherent cells

Prior to seeding the cells into 100 mm plastic culture dishes, put autoclaved microscope coverslips on the bottom of Petri dishes (four coverslips per 100 mm dish) and let the cells adhere and grow on them to the desired density.

Protocol 17.

Method

1. Place coverslips with cell side up into a plastic petri dish (—10 coverslips per dish).

2. Add 4% buffered formalin (4% formaldehyde/PBS) or Bouin's solution and incubate for 5 min.

3. Remove coverslips using forceps and dip into a container of PBS. Agitate for a few seconds in PBS.

4. Transfer for a few seconds to a second container of PBS and agitate for a few seconds.

5. Air-dry the coverslips with cell side up in a Petri containing dry Whatman filter paper.

6. Apply one or two drops of acrytol mounting medium to the bottom of the coverslip and place with cell side up on to a glass microscope slide. Let dry for 1 h or longer. If desired, each coverslip can be divided into four pieces with a diamond pencil. This permits more antibody staining conditions to be tried from a small number of cells.

7. The fixed and dried slides can be stored at 4°C for several months, or you may precede to immunostaining.

7.1.2 Suspension cells

Protocol 18.

Method

11. Take 5-10 ml cells from tissue culture or —0.2 ml of whole blood.

12. Add an equal volume of PBS, and mix.

13. Spin down at 1500 r.p.m. for 5-10 minutes (-400 g).

14. Discard the supernatant.

15. Wash once with -10 ml PBS. Then aspirate supernatant off.

16. Add 500 [aI of Bouin's solution or 4% formaldehyde/PBS.

17. Incubate at room temperature for 3-5 min.

19. Aspirate off fixative and wash 3-5 times in PBS.

10. Resuspend the cells in —0.5 ml PBS containing 0.01% NaN3 and store at 4 °C.

11. Prepare poly-L-lysine-coated slides; put one drop of solution (Sigma Chemicals, Inc.; cat. no. 1010) on the slide, smear the drop with a gloved fingertip, and let it polymerize at 37°C for 1-3 h.

12. Mix the fixed cell suspension before applying to the slides.

13. Place a drop on the coated slides and gently spread the drop with a pipette tip over an area of ~2 cm diameter. Up to three drops can be placed on the slide and spread over a larger area.

15. Store in slide boxes or proceed to immunostaining procedure.

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