Immunocytochemistry - Cell Preparation Methods

 

Immunocytochemistry Methods, Techniques and Protocols

 

ICC Main Page > Cell Preparation

 

Preparation of Coated Slides

1.1 Position clean glass slides in a staining rack

1.2 Immerse the slides for 30 min in a large staining dish containing a 1:10 dilution of 0.1% poly-L-lysine solution in deionized water.

1.3 Remove the slides and oven dry for 1 hr at 60 C.

 

Cell Film Slide Preparation for Blood and Cell Suspension

2.1 Add a drop of cells (blood, cell suspension, etc.) to the end of the coated glass slide.

2.2 Hold the beveled edge slide at a 45° angle to the plane of the coated slide and gently touching the surface of the slide, back the edge over the drop of cells so they spread within the 45° angle, the width of the slide

2.3 Slide the beveled edge slide toward the other end of the preparation slide (in the direction of the 135° angle) with a rapid uniform motion. The cells will spread out over the surface of the slide and form a film with a feathered edge, if done properly.

2.4 Allow to air dry for 30 minutes.

2.5 Immerse in cooled acetone or other fixatives to fix the cells.

2.6 Rinse slides 3x5 min in PBS.

2.7 Incubate the slides with 0.25-0.5% Triton X-100 in PBS for 10 minutes to permeabilize the membranes (Note: there is no need for a permeabilization step following acetone or methanol fixation).

2.8 Rinse 3x5 min in PBS.

2.9 Blocking endogenous peroxidase by incubating in 3% H2O2 in PBS for 10-30 minutes (Do this step only if a peroxidase marker is to be used. Omit this step if a fluorescent or AP marker is to be used).

2.10 Rinse in 3x5 min in PBS.

2.11 Blocking with 2-5% normal serum in PBS for 1 hour (Normal serum should be the same species as the secondary antibody is raised).

2.12 Incubate with primary antibody and proceed to routine immunocytochemistry procedure (see below)

 

Swabbed Slide Preparation

3.1 Obtain sample on sterile swab.

3.2 Smear the sample onto the glass slide using the majority of the surface area to distribute the specimen.

3.3 Spray fix the material with Cyto Prep.

3.4 Allow to air dry.

3.5 Postfix the slides with 10% neutral buffered formalin for 30 seconds.

3.6 Rinse 3x5 min with PBS.

3.7 Incubate the slides with 0.25-0.5% Triton X-100 in PBS for 10 minutes to permeabilize the membranes (Note: there is no need for a permeabilization step following acetone or methanol fixation).

3.8 Rinse 3x5min in PBS to remove detergents.

3.9 Blocking endogenous peroxidase by incubating in 3% H2O2 in PBS for 10-30 minutes (Do this step only if a peroxidase marker is to be used. Omit this step if a fluorescent or AP marker is to be used).

3.10 Rinse in 3x5 min in PBS.

3.11 Blocking with 2-5% normal serum in PBS for 1 hour (Normal serum should be the same species as the secondary antibody is raised).

3.12 Incubate with primary antibody and proceed to routine immunocytochemistry procedure.

 

Cell Culture Slide Preparation

4.1 Transfer 200 ul of cell culture to the wells of a chamber slide or slides of your choice (Choice of slide design is often dictated by the experiment. Some slides have four wells, some have eight, some are glass, and some are plastic. Glass is recommended simply because the slide becomes more versatile, acetone can be used as a fixative, and finished slides can be dehydrated in ethanol and cleared in xylene)

4.2 Allow cells to grow to confluence with the addition of fresh media.

4.3 Wash the cells thoroughly 5x2 min in PBS.

4.4 Fix cells with 95% ethanol, 5% glacial acetic acid for 3-5 minutes.

4.5 Rinse 5x2 min in PBS.

4.6 Incubate the slides with 0.25-0.5% Triton X-100 in PBS for 10 minutes to permeabilize the membranes (Note: there is no need for a permeabilization step following acetone or methanol fixation).

4.7 Rinse 3x5 min in PBS.

4.8 Blocking endogenous peroxidase by incubating in 3% H2O2 in PBS for 10-30 minutes (Do this step only if a peroxidase marker is to be used. Omit this step if a fluorescent or AP marker is to be used. 

4.9 Rinse in 3x5min in PBS.

4.10 Blocking with 2-5% normal serum in PBS for 1 hour (Normal serum should be the same species as the secondary antibody is raised).

4.11 Incubate with primary antibody and proceed to routine immunocytochemistry procedure.

 

Preparation of Cell Culture Block for Paraffin Embedding Using HistoGel

5.1 Microwave HistoGel (Richard Allan Scientific, Cat# HG-4000-012) on low for 5-15 seconds. Make sure to loosen the cap before heating a tube of HistoGel to prevent rupturing of the tube (An alternatively you can also place HistoGel into a boiling water bath for 3-10 minutes.

5.2 After HistoGel is liquefied, the temperature may be lowered to 50 C ± 5 and it will remain in the liquid state. A lower temperature will allow the gel to solidify more quickly after it is dispensed onto a specimen.

5.3 Centrifuge ethanol-fixed cell suspension.

5.4 Remove the supernatants from the centrifuge tube.

5.5 Add 4-6 drops of liquefied HistoGel with a pipette to cell pellet at bottom of centrifuge tube.

5.6 Either vortex specimen for several seconds to adequately and thoroughly mix cells and HistoGel together, OR allow HistoGel to settle to the bottom of tube.

5.7 Allow HistoGel to solidify by cooling to near room temperature (<20 C). This can be achieved by use a cooling plate, ice tubes, freeze pack, or allowing to cool naturally.

5.8 Remove HistoGel pellet containing the specimen and place inside a tissue cassette.

5.9 Histologically process HistoGel “button” containing the cell pellet as a standard histology specimen.

5.10 Paraffin embedding and sectioning, and then following standard immunohisotchemical staining procedure.

 

Cytospin Slide Preparation

 

6.1 Position slides in slide holders with filter cards and sample chambers and attach to cytocentrifuge rotor.

6.2 Prepare cell suspension of 500 cells/mm3 (ul) with PBS (Preparations that are too dilute for cytocentrifugation may be dropped with a pipet using the location of the filter card as a guide, and allowed to dry).

6.3 Add 0.1 ml cell suspension to chamber and centrifuge at 1000 rpm for 5 minutes (It is important to get just the right amount of speed for cytocentrifugation, as too much speed will flatten the cell, and too little will not allow the cells to adequately bind to the slide).

6.4 Remove slide and immediately dip in 95% ethanol, 5% glacial acetic acid fixative for 2 minutes.

6.5 Rinse 3x5 min with PBS to remove fixative.

6.6 Incubate the slides with 0.25-0.5% Triton X-100 in PBS for 10 minutes to permeabilize the membranes (Note: there is no need for a permeabilization step following acetone or methanol fixation).

6.7 Rinse 3x5min in PBS to remove detergents.

6.8 Blocking endogenous peroxidase by incubating in 3% H2O2 in PBS for 10-30 minutes (Do this step only if a peroxidase marker is to be used. Omit this step if a fluorescent or AP marker is to be used).

6.9 Rinse 3x5 min in PBS.

6.10 Blocking with 2-5% normal serum in PBS for 1 hour (Normal serum should be the same species as the secondary antibody is raised).

6.11 Incubate with primary antibody and proceed to routine immunocytochemistry procedure.

 

Touch Prep Slide Preparation

 

7.1 Take the refrigerated acetone and add to a Coplin jar.

7.2 Position the excised tissue directly above the slides, best side facing the slide, using forceps (The purpose for the preparation of these types of slide is to examine the cells of a tissue quickly, with no need for tissue preparation and cutting. It is important to work swiftly, as the tissue will start to deteriorate the moment it is obtained. Therefore, it is important to have all the preparations ready for the rapid handling of the excised tissue. As soon as a surface of the tissue is decided on, it should immediately be touched to a waiting slide, oriented properly and ready to be fixed).

7.3 Align the slide horizontally on flat surface.

7.4 Gently touch the selected exposed tissue surface down onto the slide (Excess tissue fluid may be absorbed with a little gauze, but avoid touching any wet cells. Also, gentle pressure is required, but if too forceful, some cells may be destroyed).

7.5 Apply slight pressure then remove the tissue after a few seconds.

7.6 With a minimum of disturbance, immerse slide into the cold acetone solution for 5-10 minutes (For touch preps, the slide should be immersed in the acetone as soon as possible, but the cells need a moment to adhere to the plane of the glass. Slowly dip the slide into the acetone, as a violent action at this point could wash off some of the cells. As is the case with frozen sections, fixation is a matter of choice. In this instance, the use of acetone is preferred because the cells are still whole, and the membranes require disruption in order for the contents to be accessible for later analysis. However, an ethanol/acid fixative - 95% ethanol, 5% glacial acetic acid, is perfectly acceptable if desired).

7.7 Allowed to air dry.

7.8 Rinse the slides 3x5 min in PBS.

7.9 Incubate the slides with 0.25-0.5% Triton X-100 in PBS for 10 minutes to permeabilize the membranes (Note: there is no need for a permeabilization step following acetone or methanol fixation).

7.10 Rinse 3x5min in PBS to remove detergents.

7.11 Blocking endogenous peroxidase by incubating in 3% H2O2 in PBS for 10-30 minutes (Do this step only if a peroxidase marker is to be used. Omit this step if a fluorescent or AP marker is to be used. 

7.12 Rinse in 3x5 min in PBS.

7.13 Blocking with 5% normal serum in PBS for 1 hour (Normal serum should be the same species as the secondary antibody is raised).

7.14 Incubate with primary antibody and proceed to routine immunocytochemistry procedure.