Materials

Cytokine-specific Primary Antibodies

• unlabeled or biotinylated antigen-affinity purified polyclonal antibodies R&D Systems 'AF or BAF series or selected monoclonal antibodies.

Secondary Antibodies and Secondary Reagents

• Biotinylated donkey anti-goat IgG (Jackson Immuno Research Lab Catalog # 705-066-147)
• Biotinylated goat anti-mouse IgG₁ Caltag Lab Catalog # M32115)
• Vectastain Elite-ABC-peroxidase (Vector Lab Catalog # PK6100)
• ExtrAvidin-HRP (Sigma Catalog # E2886)

Substrate

• 3,3'-Diaminobenzidine tetra hydrochloride (DAB) (Sigma Catalog # D5637)

Buffers and Additional Supplies

• Fixation Buffer Formaldehyde (37% v/v Sigma) is diluted in PBS to a final formaldehyde concentration of 2% (v/v) and adjust pH to 7.4. Light sensitive, store at 4°C in the dark; prepare working dilution just prior to fixation
• Wash Buffer Earls Buffered Salt Solution (EBSS) (Gibco BRL)
• Wash Buffer-Saponin EBSS with 0.1% (w/v) Saponin (Sigma Catalog # S4521) and adjust pH to 7.2-7.4 with NaOH.
• Endogenous Peroxidase Blocking Buffer 3mM NaN₃ in EBSS with 1% H₂O₂ and 0.1% (w/v) Saponin.
• Endogenous Biotin Blocking Buffer Avidin/Biotin Blocking Kit (Vector Lab Catalog # SP2001). Both Avidin D and Biotin should be supplemented with 0.1% (w/v) Saponin.
• Immunohistochemistry Mounting Medium PBS buffered glycerol 1:9 (v/v) and adjust pH to 7.4.
• Mayer's Hematoxylin Counterstain (Sigma Catalog # MHS-16)
• Saponin stock Prepare a stock of 10 % (w/v) Saponin in EBSS. Prepare fresh, since crude saponin powder batches are generally fungi infected.
• Microscope slides Adhesion slides (smeared cells); TC microscope glass slides (tissue sections); HTC slides (Cel-line Assoc. Catalog # 10-618)
• Frozen tissue embedding OCT-compound (VWR Catalog # 25608-930)
• Humidified Chamber A plastic light protected box lined with wet paper towels.

Controls

Positive staining controls

• Stain fixed, permeabilized cytokine-cDNA transfected eukaryotic cells expressing the intra-cellular target cytokine protein.
• Stain cultured blood mononuclear cells or spleen cells that have been harvested, fixed and permeabilized at the peak of cytokine production after in vitro stimulation with strong polyclonal activators such as phorbol 12-myristate 13 acetate/ionomycin, anti-CD3⁺CD28 monoclonal antibody, LPS or bacterial superantigens such as staphylococcal enterotoxin A (SEA), staphylococcal enterotoxin B (SEB) or streptococcal pyrogenic exotoxin A (SPE-A).

Negative staining controls

• Ligand blocking control: to study the specificity of the cytokine staining by a preincubation of the cytokine-specific antibody with its target cytokine at a molar ratio of 1:10 prior to addition to the cells.
• Isotype matched control immunoglobulin: Stain the cells by a primary isotype pre-matched immunoglobulin of irrelevant antigen-specificity at the same concentration as the cytokine-specific antibodies.
• Unlabeled antibody control: Cells that will be stained by a fluorochrome or a biotinylated primary cytokine-specific antibody will be incubated initially with the unconjugated version of the same antibody.
• Non transfected eukaryotic cells and unstimulated PBMNC are good negative controls.

Cytokine Detection in Tissue using Immunohistochemistry

This method is based on cryopreserved tissue, which after sectioning will be fixed in formaldehyde and stained by cytokine-specific antibodies by indirect immunoenzymatically based technique. It has proven mandatory to include saponin in all antibody incubations, despite the fact that the tissue has been cryostat-sectioned.

Sample preparation and fixation

1. Immediately snap freeze fresh tissue in isopentane in dry ice and kept at -70°C. Do not allow frozen tissue to thaw before cutting.
2. Embed tissue completely in OCT compound prior to cryostat sectioning.
3. Cut cryostat sections at 5-10 µm and mount on HTC glass slides.Note: Suggested cryostat temperature is -23°C and the tissue specimen is -18°C. The section will curl if the specimen is too cold and if it is too warm it will stick to the knife.
4. Air dry the sections for 30 minutes at room temperature to prevent sections from falling off the slides during antibody incubations.Note: Store the slides unfixed for several months at -70°C. Frozen tissue samples for analysis at later time points should be stored unsectioned.
5. Immediately add 50 µL of ice-cold Fixation buffer on each tissue section upon removal from the freezer.
6. Fix for 8 minutes at 4°C or less optimally at 20°C for 20 minutes.

Antibody Incubations

1. Wash samples three times with Wash Buffer-Saponin for three minutes each.
2. Block endogenous peroxidase activity in the specimens with Endogenous Peroxidase Blocking Buffer for 30-60 minutes at room temperature in the dark.
3. Wash three times with Wash Buffer-Saponin for three minutes each.
4. Block endogenous biotin with Endogenous Biotin Blocking Buffer for 30 minutes in a sequential mode. Prepare both the Avidin D and the Biotin by adding 6 drops/mL of the stock solution to Wash Buffer-Saponin. Note: Incubation with avidin D may need to be prolonged in tissue with high endogenous biotin activity. The diluted biotin should be supplemented with 2% serum from the same species as the source of the secondary antibody.
5. Wash sample twice with Wash Buffer-Saponin.
6. Incubate overnight (at least 12 hours) with 50-100 µL of primary antibody (0.5-5.0 µg/mL in Wash Buffer-Saponin) or negative control antibody in a humidified chamber at room temperature. It is essential that the specimens will not dry out.
7. Wash samples three times with Wash Buffer-Saponin for three minutes each.
8. Incubate for 30-45 minutes at room temperature with 25-50 µL of a biotinylated secondary antibody (either biotin-donkey anti-goat IgG diluted 1:700 or biotin-goat anti-mouse IgG₁ or IgG_2A or IgG_2B diluted 1:500) in Wash Buffer-Saponin.
9. Wash slides three times in Wash-Buffer-Saponin for 30 seconds each.
10. Prepare Vectastain Elite ABC-peroxidase reagent according to manufacturers' instructions supplemented with saponin (0.1% w/v) 30 minutes prior to incubation.
11. Incubate with Vectastain Elite ABC-peroxidase for 30 minutes at room temperature in the dark.
12. Wash samples three times with Wash Buffer without saponin for three minutes prior to substrate incubation.
13. Prepare the substrate according to manufacturers instructions without any supplementation of saponin.
14. Incubate sample for approximately 8 minutes (monitor in the microscope). A brown color reaction with distinct morphology is developed with DAB in the peroxidase system.
15. Stop the development of the color reaction by repeated washes in distilled water.
16. Counterstain, if desired, with hematoxylin for 1-5 seconds. Slides are then air dried and can be mounted and coverslipped with Immunohistochemistry Mounting Medium without any further dehydration. Slides that need to be kept for long periods in optimal shape can be dehydrated in ethanol and mounted in special mounting medium.

Immunostaining of individual cytokine-producing cells smeared on slides

Suitable for use on single-cell suspensions from peripheral blood, lymphoid tissue or cultured cell-lines.

Sample Preparation and Fixation

1. Harvest cells and wash twice in V-bottomed tubes with cold Wash Buffer by centrifugation (400 x g for 5 minutes) to remove extracellular proteins, including cytokines.
2. Resuspend to 1-5 x 10⁶ cells/mL in Wash Buffer.
3. Transfer 10-15 µL of the cell suspension to each reaction field on the adhesion slide.
4. Allow the cells to adhere electrostatically in a monolayer for 10 minutes at room temperature in the humidified chamber to prevent the cells from drying out.
5. Add 50 µL of ice-cold Fixation Buffer to each field to fix the cells.
6. Incubate for 20 minutes at 4°C.
7. Wash three times with Wash Buffer to remove free formaldehyde.
8. Add 25 µL of 2% fetal bovine serum in Wash Buffer to block unbound surface area on the slide.
9. Incubate for 10 minutes at 37°C.
   1. Wash slides three times in Wash Buffer-Saponin. The slides are now ready for staining.
   2. Alternatively, wash the slides in Wash Buffer and allow the slides to dry. Dried slides can be stored at -20°C several months before being stained. Prior to staining the slides should be washed in Wash Buffer-Saponin.

Antibody Incubation

All antibody incubations and washes are performed in Wash Buffer-Saponin to keep the cells permeable for antibodies to penetrate the cell membranes.

Detection using Biotin-labeled antibodies

1. Incubate in Endogenous Peroxidase Blocking Buffer for 30 minutes at room temperature in the dark to block endogenous peroxidase activity in the cells (this step can be omitted if cells are to be stained by fluorochromes or non-peroxidase based enzymatic methods).
2. Block endogenous biotin activity with the Avidin/Biotin blocking kit in a two step procedure for 30 minutes in the presence of saponin, described in steps 3-5.
3. Incubate each cell spot on slides with Avidin for 15 minutes supplemented with saponin (0.1% w/v).
4. Wash each cell spot on slides twice in Wash Buffer-Saponin.
5. Incubate in Biotin for 15 minutes supplemented with saponin (0.1% w/v).
6. Wash each cell spot on slides twice in Wash Buffer-Saponin.
7. Incubate each cell spot on slides for 30 minutes at room temperature with 15 µL unlabeled or biotinylated cytokine-specific antibodies (0.5-5 µg/mL) diluted in Wash Buffer-Saponin.
8. Wash slides three times in Wash Buffer-Saponin. Note: If using R&D Systems biotinylated antibody skip steps 9 and 10 and continue.
9. Incubate each cell spot on slides for 30 minutes at room temperature with 15 µL of a biotinylated secondary antibody (either biotin-donkey anti-goat IgG Fab₂ diluted 1:700; or biotin-goat anti mouse IgG₁ or IgG_2A or IgG_2B diluted 1:500) in Wash Buffer-Saponin.
10. Wash slides three times in Wash Buffer-Saponin.

Cytokine-specific staining based on either biotinylated primary antibodies or unlabeled primary antibodies along with biotinylated secondary antibodies can then be developed by techniques based on immunoflourescence or immunoenzymatic methods.

Immunoenzymatic Technique

1. Prepare Vectastain Elite ABC-peroxidase reagent according to manufacturers' instructions and supplement with saponin (0.1% w/v), 30 minutes prior to step 2.
2. Incubate for 30 minutes with Vectastain Elite ABC-peroxidase at room temperature in the dark.
3. Wash slides three times with distilled water prior to substrate incubation.
4. Prepare the substrate according to the the protocol given by the manufacturer without any supplementation of saponin.
5. Incubate for approximately 8 minutes (monitor in the microscope) at room temperature in the dark. A brown color reaction with distinct morphology is developed with DAB in the peroxidase system.
6. Stop the development of the color reaction by repeated washes in distilled water.
7. Counterstain, if desired, with hematoxylin for 1-5 seconds. Slides are then air dried and coverslipped with Immunohistochemistry Mounting Medium. Slides that need to be kept for long periods in optimal shape should be dehydrated in ethanol and mounted in special mounting medium.

Frequently Asked Questions

Question: Are background signals caused by the developing substrate?
Test: Incubate with substrate alone.
Cause of Background: Endogenous enzyme (peroxidase/alkaline phosphatase) activity in sample
Remedy: Block with Endogenous Peroxidase Blocking Buffer (with Levamisol in the alkaline phosphate system).

Question: Are background signals caused by endogenous biotin activity in the sample?
Test: (Block if necessary as in Question 1) Incubate with Vectastain ABC (or ExtraAvidin HRP/AP) and incubate with substrate.
Cause of background: Endogenous biotin activity.
Remedy: Block with Endogenous Biotin/Avidin Blocking-Buffer.

Question: Does the secondary biotinylated antibody cause background signals?
Test: (Block if necessary as in Question 1 and/or Question 2). Incubate with the following three steps: secondary antibody; Vectastain ABC-kit (or ExtrAvidin HRP/AP); and substrate.
Cause of background: Fc-receptor binding of secondary antibody used at too high concentration.
Remedy: Block with species matched serum to secondary antibody or try to dilute the secondary antibody.

Question: Does the primary cytokine specific antibody generate background signals?
Test: (Block if necessary as in Question 1 and/or Question 2). Use secondary antibody at optimal concentration. Incubate with the following: primary antibody; secondary antibody; Vectastain ABC (or ExtrAvidin HRP/AP); and with substrate.
Cause of background: Cytokine-detection antibody used at too high concentration or unsuitable for immunostaining.
Remedy: Titrate antibody or try another cytokine detecting antibody.

Question: Is the cytokine staining specific?
Test: Incubate cytokine-detecting antibody with target cytokine overnight and finally add 0.1 (saponin prior to staining as in Question 4).
Cause of Background: Cytokine-detecting antibody used at too high concentration or unsuitable for immunostaining.
Remedy: Titrate antibody or try another cytokine detecting antibody.

Technical Hints

Permeabilization: It is crucial that saponin is present during all antibody incubations and washes to make the staining procedure successful. For detection of intracellular cytokines, the cytokine-specific antibodies must penetrate through the cell surface membrane, the cytosol, the membranes of the endoplasmic reticulum and the Golgi organelle. The detergent, saponin has been shown to intercalate in the membranes to replace cholesterol and to permeabilize cells in a reversible way, maintaining much of the morphology of the membrane structure of the cell.
Fixation: A solution of phosphate-buffered formaldehyde has been found to preserve cell morphology as well as surface and intracellular antigenicity with minuscule cell aggregation and cell loss. Only fixed cells will stand the effects of detergent treatment.
Controls: Evidence for specificity of the cytokine staining should be based on parallel studies of isotype controls, staining with the secondary antibodies alone and an abolishment of immunoreactivity by preabsorption of the cytokine-specific antibody with the corresponding cytokine protein.
Stimulation of PBMNC for cytokine production: The strongest and most diversified cytokine production is seen after PMA-ionomycin activation of Ficoll separated PBMNC. Both monocytes and lymphocytes will be activated. The cells are co-cultivated with PMA (1 ng/mL), ionomycin (500 nM) and harvested after 4 hours. After 4 hours, one can expect to see IL-1 alpha, IL-1 beta, IL-1ra, IL-2, IL-3, for various periods. If cells are harvested after 3 hours, one can find monokines such as IL-1 alpha, IL-1 beta, IL-1ra, IL-6, IL-8, MIP-1 alpha, MIP-1 beta, and TNF-alpha. However, cytokines produced by lymphocytes, IL-12 or substantial IL-10 production is not found in the cultures.

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Our thanks to Drs. Ulf Andersson, Ann-Charlotte Aveberger, Lars Björk, Caroline Ekberg, Thomas Fehniger, Mark Litton, Lena Radler, Birgitta Sander, Ulrika Skansén-Saphir, Ann-Kristin Ulfgren, Karin Ågren, Karin-Åkerlund (Department of Immunology, Stockholm University) for the submission of the protocol and revision of the manuscript.