Cytokine Immunocytochemistry Fax (65) 860-1590 Fax (49) 40 531 58 92 Fax (81) 3 541-381-55 E-Mail: [email protected]

Cytokine Immunocytochemistry Fax (65) 860-1590 Fax (49) 40 531 58 92 Fax (81) 3 541-381-55 E-Mail: Canada@Pharmingen.Com

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99-6081-74A Technical Assistance 1-800-TALK-TEC (825-5832) • Fax 619-812-8888 World Wide Web http://www.pharmingen.comXX Table of Contents Introduction The term cytokines refers to a broad group of signaling proteins that, in general, Introduction ...... 1 are produced transiently after cellular activation, act locally as autocrine, juxtacrine Immunocytochemical Measurements of Mouse Cytokines ...... 2 or paracrine biological response modifiers, and exert their actions by binding to specific high-affinity receptors on target cells. The vast cytokine group includes Immunocytochemical Measurements of Human Cytokines . . . . . 3 members of the interleukin, interferon, tumor necrosis factor, colony stimulating factor, transforming growth factor and chemokine families of proteins. Due to the Important Points to Consider when Choosing a critical roles played by cytokines in the regulation of immunity and inflammation, Method to Study Cytokine Production by Individual Cells ...... 4 there has been increased emphasis on defining the qualitative, quantitative, temporal and spatial patterns of their expression. These types of analyses have Comparison of Cytokine Immunocytochemistry and Flow benefited tremendously from the recent introduction of methods that allow for the Cytometry ...... 5 characterization of cytokine-producing cells at the single cell level. Such methods are often based on the use of monoclonal antibodies that are specific for a particular Relative Sensitivities of Immunocytochemistry cytokine and immunofluorescent or immunoenzymatic techniques to detect and and Flow Cytometry ...... 6 quantify specific antibody binding to target cytokines (Sander et al., 1991). In the latter case, immunocytochemistry (ICC) can be a powerful immunoenzymatic Critical Parameters in Immunocytochemistry ...... 7 technique that allows for the detection of the cytokines associated with individual cells that have been immobilized on microscopic slides. Schematic of Staining Protocol ...... 8-9 An important goal for PharMingen has been to develop and to provide high Analysis of Mouse TNF-α-Producing Cells quality antibodies that can be used to study cytokine expression at the single cell by Immunocytochemistry ...... 10 level. In this mailer, PharMingen is pleased to provide a list of selected antibody clones that are now available for the immunocytochemical staining of human Features of Intracellular Staining ...... 11 and mouse cytokine-producing cells. Moreover, reagents and methods are presented that are suitable for the immunoenzymatic analysis of cytokine Comparison of Immunocytochemical Detection Systems ...... 11 protein expression using the technique of immunocytochemistry.

Cytokine Immunocytochemistry Protocol ...... 12-13

Product Listing ...... 14

Controls for Cytokine Immunocytochemistry ...... 15

Cytokine Immunocytochemistry Program Overview ...... 16

References ...... 16

We are thankful to Dr. Jan Andersson (Karolinska Institute, Sweden) and Dr. Thomas E. Fehniger (Astra Draco, Sweden) for consulting with PharMingen in the development of methods and reagents for the immunocytochemical analysis of cytokine-producing cells.

Cover image is an artistic rendering of cytokine immunocytochemical staining and does not represent actual data. All products are for research use only. Not for use in diagnostic or therapeutic procedures.

Orders 1-800-848-MABS (6227)• Phone 619-812-8800 Technical Assistance 1-800-TALK-TEC (825-5832) • Fax 619-812-8888 XXi World Wide Web http://www.pharmingen.com World Wide Web http://www.pharmingen.comXX1 Table of Contents Introduction The term cytokines refers to a broad group of signaling proteins that, in general, Introduction ...... 1 are produced transiently after cellular activation, act locally as autocrine, juxtacrine Immunocytochemical Measurements of Mouse Cytokines ...... 2 or paracrine biological response modifiers, and exert their actions by binding to specific high-affinity receptors on target cells. The vast cytokine group includes Immunocytochemical Measurements of Human Cytokines . . . . . 3 members of the interleukin, interferon, tumor necrosis factor, colony stimulating factor, transforming growth factor and chemokine families of proteins. Due to the Important Points to Consider when Choosing a critical roles played by cytokines in the regulation of immunity and inflammation, Method to Study Cytokine Production by Individual Cells ...... 4 there has been increased emphasis on defining the qualitative, quantitative, temporal and spatial patterns of their expression. These types of analyses have Comparison of Cytokine Immunocytochemistry and Flow benefited tremendously from the recent introduction of methods that allow for the Cytometry ...... 5 characterization of cytokine-producing cells at the single cell level. Such methods are often based on the use of monoclonal antibodies that are specific for a particular Relative Sensitivities of Immunocytochemistry cytokine and immunofluorescent or immunoenzymatic techniques to detect and and Flow Cytometry ...... 6 quantify specific antibody binding to target cytokines (Sander et al., 1991). In the latter case, immunocytochemistry (ICC) can be a powerful immunoenzymatic Critical Parameters in Immunocytochemistry ...... 7 technique that allows for the detection of the cytokines associated with individual cells that have been immobilized on microscopic slides. Schematic of Staining Protocol ...... 8-9 An important goal for PharMingen has been to develop and to provide high Analysis of Mouse TNF-α-Producing Cells quality antibodies that can be used to study cytokine expression at the single cell by Immunocytochemistry ...... 10 level. In this mailer, PharMingen is pleased to provide a list of selected antibody clones that are now available for the immunocytochemical staining of human Features of Intracellular Staining ...... 11 and mouse cytokine-producing cells. Moreover, reagents and methods are presented that are suitable for the immunoenzymatic analysis of cytokine Comparison of Immunocytochemical Detection Systems ...... 11 protein expression using the technique of immunocytochemistry.

Cytokine Immunocytochemistry Protocol ...... 12-13

Product Listing ...... 14

Controls for Cytokine Immunocytochemistry ...... 15

Cytokine Immunocytochemistry Program Overview ...... 16

References ...... 16

Orders 1-800-848-MABS (6227)• Phone 619-812-8800 Technical Assistance 1-800-TALK-TEC (825-5832) • Fax 619-812-8888 XXi World Wide Web http://www.pharmingen.com World Wide Web http://www.pharmingen.comXX1 Immunocytochemical Measurements of Mouse Cytokines Immunocytochemical Measurements of Human Cytokines

(A) Mouse IL-2 (Clone JES6-1A12) (B) Mouse IFN-γ (Clone XMG1.2) (H) Human IL-6 (Clone MQ2-6A3) (I) Human IL-2 (Clone MQ1-17H2)

(C) Mouse TNF-α (Clone MP6-XT22) (D) Mouse IL-3 (Clone MP2-8F8) (J) Human IL-5 (Clone JES1-39D10) (K) Human IL-13 (Clone JES10-5A2)

(E) Mouse IL-4 (Clone 11B11) (F) Mouse IL-10 (Clone JES5-16E3) (L) Human GM-CSF (Clone BVD2-21C11) (M) Human TNF-α (Clone MAB11)

Figure 1. Immunocytochemical detection of cytokine- producing cells using PharMingen’s antibodies directed against mouse and human cytokines. Human PBMC were isolated by Lymphoprep (Nycomed) density gradient centrifugation and were stimulated with LPS (1 µg/ml, Sigma) overnight in the presence of Mouse splenocytes were RBC-lysed, washed and cultured with GolgiStopª that contains the protein transport inhibitor, monensin (Cat. No. 2092KZ). Cells were stained for human IL-6 (MQ2-6A3, H). Alternatively human PBMC PMA (5 ng/ml, Sigma) and ionomycin (500 ng/ml, Sigma) for 4 hr were cultured for 2 days with plate bound anti-CD3 and soluble anti-mouse CD28 in the presence of recombinant IL-2 and recombinant IL-4. The cells were subse- at 37¡C with GolgiPlugª (Cat. No. 2301KZ) that contains the pro- quently harvested, washed and recultured with recombinant IL-2 and recombinant IL-4 for an additional 3 days. Finally, the cells were harvested, washed and cultured tein transport inhibitor, brefeldin A. Cells were harvested (4 hr) with PMA and ionomycin in the presence of GolgiStopª (Cat. No. 2092KZ). Cells were harvested and stained by ICC for human IL-2 (MQ1-17H2, I), human and stained by immunocytochemistry (ICC) for mouse IL-2 IL-5 (JES1-39D10, J), human IL-13 (JES10-5A2, K), human GM-CSF (BVD2-21C11, L), and human TNF-α (MAB11, M). The specificity of staining was confirmed by (JES6-1A12, A), IFN-γ (XMG1.2, B), and TNF-α (MP6-XT22, C). using the appropriate immunoglobulin isotype and ligand-blocking controls (A-M; Nomarski optics; original magnification 400 X). Alternatively, the RBC-lysed splenocytes were enriched for CD4+ cells and were cultured for 2 days with plate bound anti-mouse CD3 and soluble anti- mouse CD28 in the presence of recombinant IL-2 and recombinant IL-4. The cells were subsequently harvested, washed and recultured with recombinant IL-2 and recombinant IL- 4 for an additional 3 days. Finally, the cells were harvested, washed and cultured (4 hr) with PMA and ionomycin in the presence of GolgiPlugª (Cat. No. 2301KZ). Cells were harvested and stained by ICC for mouse IL-3 (MP2-8F8, D), IL-4 (11B11, E), IL-10 (JES5-16E3, F) and GM-CSF (MP1-22E9, G). (G) Mouse GM-CSF (Clone MP1-22E9)

Orders 1-800-848-MABS (6227)• Phone 619-812-8800 Technical Assistance 1-800-TALK-TEC (825-5832) • Fax 619-812-8888 2 World Wide Web http://www.pharmingen.com World Wide Web http://www.pharmingen.com3 Immunocytochemical Measurements of Mouse Cytokines Immunocytochemical Measurements of Human Cytokines

(A) Mouse IL-2 (Clone JES6-1A12) (B) Mouse IFN-γ (Clone XMG1.2) (H) Human IL-6 (Clone MQ2-6A3) (I) Human IL-2 (Clone MQ1-17H2)

(C) Mouse TNF-α (Clone MP6-XT22) (D) Mouse IL-3 (Clone MP2-8F8) (J) Human IL-5 (Clone JES1-39D10) (K) Human IL-13 (Clone JES10-5A2)

(E) Mouse IL-4 (Clone 11B11) (F) Mouse IL-10 (Clone JES5-16E3) (L) Human GM-CSF (Clone BVD2-21C11) (M) Human TNF-α (Clone MAB11)

Orders 1-800-848-MABS (6227)• Phone 619-812-8800 Technical Assistance 1-800-TALK-TEC (825-5832) • Fax 619-812-8888 2 World Wide Web http://www.pharmingen.com World Wide Web http://www.pharmingen.com3 Important points to consider when Comparison of Cytokine Immunocytochemistry choosing a method to study cytokine and Flow Cytometry Cytokine Cytokine production by individual cells. Immunocytochemistry Flow Cytometry

The method of choice for studying cytokine production at the single cell level is Sensitivity •High •High strictly dependent upon the experimental model system being used, the available facilities and equipment, and the types of experimental questions that one seeks to •Enzyme-amplified antibody •Fluorescent antibody-based answer (see table). Presently, the most widely used method to characterize the Detection detection system Sensitive electronic detection nature of individual cytokine-producing cells has been immunofluorescent staining System and amplification of fluorescent coupled with flow cytometric analysis. This constitutes a rapid and highly sensitive antibody signals technique that offers the capacity for multiparameter characterization of individual cytokine-producing cells. A potential drawback of this technique is that it requires Features •Detects cytokine production at •Detects cytokine production access to a flow cytometer and a computer with appropriate software for data the single cell level at the single cell level •Rapid assay •Rapid assay acquisition and analysis. Immunocytochemistry, on the other hand, achieves a •Simultaneous morphological and •Multiparameter analysis of similar sensitivity when compared with flow cytometry-based methods by cytokine staining analysis: physical and fluorescent staining employing a straightforward enzyme-amplified detection system (Figure 2, page 6). enables distinction of intracellular characteristics of cytokine- versus cell surface- bound producing cells As a result, immunocytochemical results can be acquired with the use of an ordinary cytokines light microscope that is common to most research laboratories. In addition, the •Processed samples can be stored •Processed samples can be processed specimens can be stored without loss of quality and can be reexamined for long periods without loss of stored up to 1 wk at 4°C quality as needed for an extended period of time. Moreover, because individual cell •Requires a light microscope for •Requires a flow cytometer for morphology can be visualized by using this method, one can potentially identify analysis analysis different cell types as well as distinguish between cytokine-producing cells and cells Considerations •Finite number of cells can be •Large number of cells can that stain positively due to receptor-mediated cytokine binding (Gordon, 1991). visualized in each optical field be acquired and analyzed •Image analysis equipment and •Flow cytometer enables An important consideration when performing immunocytochemical studies is that appropriate software can be used quantitation of cytokine the number of cells that can be observed in each optical field of a microscopic slide for quantitation of cytokine staining signals is finite. Thus, in order to identify significant numbers of cytokine-producing cells, staining signals one may need to observe numerous optical fields. This may be problematic in cases where the number of cytokine-producing cells may be very low (less than 1%). For example, when the investigator is interested in studying antigen-specific responses, it may be difficult to count statistically-significant numbers of cytokine-positive cells (Suni et al. 1998). An additional consideration is that while flow cytometers routinely quantify the signal, similar measurements with immunocytochemistry require additional image analysis equipment (Björk et al. 1996). Despite these concerns for many types of experimental systems, immunocytochemistry constitutes an extremely powerful technique for enumerating and characterizing cytokine- producing cells.

Orders 1-800-848-MABS (6227)• Phone 619-812-8800 Technical Assistance 1-800-TALK-TEC (825-5832) • Fax 619-812-8888 XX4 World Wide Web http://www.pharmingen.com World Wide Web http://www.pharmingen.comXX5 Important points to consider when Comparison of Cytokine Immunocytochemistry choosing a method to study cytokine and Flow Cytometry Cytokine Cytokine production by individual cells. Immunocytochemistry Flow Cytometry

Orders 1-800-848-MABS (6227)• Phone 619-812-8800 Technical Assistance 1-800-TALK-TEC (825-5832) • Fax 619-812-8888 XX4 World Wide Web http://www.pharmingen.com World Wide Web http://www.pharmingen.comXX5 Relative Sensitivities of Immunocytochemistry and Flow Cytometry Critical Parameters in Immunocytochemistry 1. Fixation: The proper fixation of cells is a very critical step for performing immunocytochemical analyses. In this regard, formaldehyde has been shown to be a gentle fixation agent that preserves cellular morphology and 12% size (and cytokine antigenicity) without cell loss or aggregation (Sander et al., 1991). 2. Permeabilization: Amongst a number of detergents and organic solvents used for cell permeabilization (including acetone, ethanol, methanol,

Relative Cell Number n-octyl-beta-D-glucopyranoside, saponin and Triton-X), saponin was shown GM-CSF (PE) to be the best choice for intracellular cytokine staining (Sander et al., 1991, Björk., 1995). Saponin is a plant derivative that has high affinity for cholesterol. It is suggested that during permeabilization, saponin (A) (B) intercalates into the membrane and replaces cholesterol (Willingham & Pastan, 1985). Because this is a reversible process, it is important that saponin is constantly present in all staining buffers during incubations of cells with antibodies (Willingham & Pastan, 1985; Sander et al., 1991). 3. Protein transport inhibitors: Immunocytochemistry can be used to examine cells activated in the absence of protein transport inhibitors due to the high sensitivity achieved with enzyme-amplified detection systems 77% (Figure 3, page 10). This allows for cells to be stained ex vivo for intracellular cytokines without the need for in vitro restimulation. Alternatively, the investigator can apply protein transport inhibitors [e.g., GolgiStop™ containing monensin (Cat. No. 2092KZ) or GolgiPlug™ containing brefeldin A (Cat. No. 2301KZ)] to cells during stimulatory cultures to Relative Cell Number augment the detection of cytokine-producing cells caused by the GM-CSF (PE) intracellular accumulation of cytokine proteins. 4. Detection system: Like other immunoenzymatic techniques, the sensitivity (C) (D) of the immunocytochemical staining technique for detecting cytokine- producing cells is dependent upon the enzyme-substrate system used. The preferred enzyme for cytokine detection has been horseradish peroxidase Figure 2. Detection of GM-CSF-producing cells by ICC and light microscopic analysis with a sensitivity comparable to that of immunofluorescent staining and flow cytometry. Human PBMC were isolated by Lymphoprep (Nycomed) density gradient centrifugation and were stimulated overnight with PMA and because it gives strong signals and a non-diffuse staining pattern (Figure 4, ionomycin in the presence of GolgiStopª that contains the protein transport inhibitor, monensin (Cat. No. 2092KZ). Cells were divided in two aliquots. The first page 11). For high-sensitivity detection of cytokine-producing cells within aliquot was stained for human GM-CSF (BVD2-21C11) by ICC and examined by light microscopy (A) and the second aliquot was tested by immunofluorescent staining and flow cytometry (B). Alternatively human PBMC cells were cultured for 2 days with plate bound anti-CD3 and soluble anti-mouse CD28 in the presence cell populations, the use of the Avidin:Biotinylated Complex procedure of recombinant IL-2 and recombinant IL-4. The cells were subsequently harvested, washed and recultured with recombinant IL-2 and recombinant IL-4 for an addi- (ABC method) (Hsu et al., 1981a, Hsu et al., 1981b) is recommended. The tional 3 days. Finally, the cells were harvested, washed and cultured (4 hr) with PMA and ionomycin in the presence of GolgiStopª (Cat. No. 2092KZ). Cells were divided in two aliquots. The first aliquot was stained for human GM-CSF (BVD2-21C11) by ICC and examined by light microscopy (C) ABC method employs biotinylated secondary antibodies and preformed and the second aliquot was tested by immunofluorescent staining and flow cytometry (D), (A and C Nomarski optics; original magnification, 400 X). Specificity of Avidin:Biotinylated enzyme Complexes as a detection system. This staining was confirmed by using the appropriate immunoglobulin isotype and ligand-blocking controls. The two different activation protocols resulted in different lev- els of GM-CSF producing cells that were detectable with both flow cytometry and immunocytochemistry. The percentage of cytokine producing cells by ICC was procedure appears to be very sensitive and to give low background staining estimated by enumeration of at least 200 cells by light microscopy. The percentages of GM-CSF producing cells was 11% (A) and 70% (C). signals because the amount of the primary antibody required for detection of intracellular cytokines can be very small. As a result of our in-house evaluations, we recommend an ICC detection method that utilizes Avidin:Biotinylated horseradish peroxidase complexes. 5. Cytokine specific antibodies: The choice of a particular cytokine-specific antibody is very critical. High affinity, monoclonal anti-cytokine antibodies are recommended for immunocytochemical staining, as opposed to polyclonal antibody preparations, because of their capacity to target strong, highly-specific signals with correspondingly low background staining. In addition, the anti-cytokine antibody must have the capacity to recognize cytokine proteins after cell fixation with formaldehyde and permeabilization with saponin. The same antibody clones presented in this mailer have not been evaluated in ICC using other methods.

Orders 1-800-848-MABS (6227)• Phone 619-812-8800 Technical Assistance 1-800-TALK-TEC (825-5832) • Fax 619-812-8888 XX6 World Wide Web http://www.pharmingen.com World Wide Web http://www.pharmingen.comXX7 Relative Sensitivities of Immunocytochemistry and Flow Cytometry Critical Parameters in Immunocytochemistry 1. Fixation: The proper fixation of cells is a very critical step for performing immunocytochemical analyses. In this regard, formaldehyde has been shown to be a gentle fixation agent that preserves cellular morphology and 12% size (and cytokine antigenicity) without cell loss or aggregation (Sander et al., 1991). 2. Permeabilization: Amongst a number of detergents and organic solvents used for cell permeabilization (including acetone, ethanol, methanol,

Orders 1-800-848-MABS (6227)• Phone 619-812-8800 Technical Assistance 1-800-TALK-TEC (825-5832) • Fax 619-812-8888 XX6 World Wide Web http://www.pharmingen.com World Wide Web http://www.pharmingen.comXX7 Intracellular Cytokine Immunocytochemistry - Overview of Staining Protocol Block endogenous 6 biotin biotin 1 Load cells on adhesion 30 block slides endogenous 20 biotin

10 Wash 2x

Incubate slides with 7 primary Ab 60 cytokine

Fix adherent cells 2 cytokine 15 specific antibody

10 Wash 2x

Add biotinylated 10 Wash 2x 8 Secondary Ab

30 Block with 1% (w/v) 3 BSA-PBS Slides can be stored at -80˚C indefinitely secondary -80˚C before the staining antibody 30 procedure 10 Wash 2x BSA Add ABC solution 10 9 Wash 2x 4 Block with goat serum 30 in buffer containing ABC saponin Goat Ig complex block 30 saponin 10 Wash 2x

Fc receptor Add DAB substrate 10 <5 10 Wash 2x

Block endogenous 5 peroxidase 10 DAB peroxidase substrate block Wash endogenous peroxidase 11 Mount slides 10 Wash 2x

Examine slides by microscopy

Orders 1-800-848-MABS (6227)• Phone 619-812-8800 Technical Assistance 1-800-TALK-TEC (825-5832) • Fax 619-812-8888 XX8 World Wide Web http://www.pharmingen.com World Wide Web http://www.pharmingen.comXX9 Intracellular Cytokine Immunocytochemistry - Overview of Staining Protocol Block endogenous 6 biotin biotin 1 Load cells on adhesion 30 block slides endogenous 20 biotin

10 Wash 2x

Incubate slides with 7 primary Ab 60 cytokine

Fix adherent cells 2 cytokine 15 specific antibody

10 Wash 2x

Add biotinylated 10 Wash 2x 8 Secondary Ab

Fc receptor Add DAB substrate 10 <5 10 Wash 2x

Block endogenous 5 peroxidase 10 DAB peroxidase substrate block Wash endogenous peroxidase 11 Mount slides 10 Wash 2x

Examine slides by microscopy

Orders 1-800-848-MABS (6227)• Phone 619-812-8800 Technical Assistance 1-800-TALK-TEC (825-5832) • Fax 619-812-8888 XX8 World Wide Web http://www.pharmingen.com World Wide Web http://www.pharmingen.comXX9 Analysis of Mouse TNF-α-Producing Cells by Immunocytochemistry Features of Intracellular Staining The majority of cytokines have N-terminal signal sequences that allow them to follow the classical pathway of protein secretion. The classical pathway of protein secretion involves accumulation of the protein in the lumen of the endoplasmic reticulum and subsequent transfer to the Golgi compartment (Walter et al., 1994). The accumulation of cytokines in the Golgi prior to their secretion can be visualized under the microscope as perinuclear staining. Such staining is very characteristic for the cytokine-producing cells and can be very easily distinguished from extracellular binding to the plasma membrane (Figure 3). Members of the interleukin-1 family, such as IL-1α and IL-1β, that lack N-terminal signal sequences, appear to follow an alternative pathway of secretion that does not involve the Golgi apparatus (Stevenson et al., 1992). These cells are characterized by a rather diffuse staining throughout the cytoplasm (Andersson, 1992; Björk, 1995).

(A) Brefeldin A (GolgiPlug™) (B) Monensin (GolgiStop™) Comparison of Immunocytochemical Detection Systems

(A) Alkaline Phosphatase (AKP) Figure 3. Immunocytochemical identification of TNF-α-producing cells in cultures with or without protein transport inhibitors. BALB/c splenocytes were stimulated with PMA (5 ng/ml, Sigma) and ionomycin (500 ng/ml, Sigma) for 4 hr in the presence of GolgiStopª (Cat. No. 2092KZ) (B), GolgiPlugª (Cat. No. 2301KZ) (A), or no protein transport inhibitor (C). Cells were stained with anti-mouse TNF-α (MP6-XT22) (A-C) or rat IgG1 (R3-34) (data not shown). Pre incubation of anti-mouse TNF-α antibody with purified recombinant mouse TNF-α protein (Cat. No 19321T) completely blocked the staining of MP6-XT22 (D), (A-D; original magnification, 400 X).

(B) Horseradish Peroxidase (HRP)

Figure 4. Horseradish Peroxidase is the optimal detection system for cytokine immunocytochemistry. Human PBMC were isolated by Lymphoprep (Nycomed) density gradient centrifugation and were stimulated overnight with PMA and ionomycin in the presence of GolgiStopª (Cat. No. 2092KZ). Cells were stained for human GM-CSF (BVD2-21C11) by ICC (see arrows) using an AKP- (A) or an HRP- (B) based detection system. The specificity of staining was confirmed by using the appropriate immunoglobulin isotype and ligand blocking controls (A and B; Nomarski optics, original magnification, 400 X).

Orders 1-800-848-MABS (6227)• Phone 619-812-8800 Technical Assistance 1-800-TALK-TEC (825-5832) • Fax 619-812-8888 10XXWorld Wide Web http://www.pharmingen.com World Wide Web http://www.pharmingen.com11XX Analysis of Mouse TNF-α-Producing Cells by Immunocytochemistry Features of Intracellular Staining The majority of cytokines have N-terminal signal sequences that allow them to follow the classical pathway of protein secretion. The classical pathway of protein secretion involves accumulation of the protein in the lumen of the endoplasmic reticulum and subsequent transfer to the Golgi compartment (Walter et al., 1994). The accumulation of cytokines in the Golgi prior to their secretion can be visualized under the microscope as perinuclear staining. Such staining is very characteristic for the cytokine-producing cells and can be very easily distinguished from extracellular binding to the plasma membrane (Figure 3). Members of the interleukin-1 family, such as IL-1α and IL-1β, that lack N-terminal signal sequences, appear to follow an alternative pathway of secretion that does not involve the Golgi apparatus (Stevenson et al., 1992). These cells are characterized by a rather diffuse staining throughout the cytoplasm (Andersson, 1992; Björk, 1995).

(A) Brefeldin A (GolgiPlug™) (B) Monensin (GolgiStop™) Comparison of Immunocytochemical Detection Systems

(B) Horseradish Peroxidase (HRP)

Orders 1-800-848-MABS (6227)• Phone 619-812-8800 Technical Assistance 1-800-TALK-TEC (825-5832) • Fax 619-812-8888 10XXWorld Wide Web http://www.pharmingen.com World Wide Web http://www.pharmingen.com11XX Cytokine Immunocytochemistry Protocol 15. Wash 2X in PBS with 5 min incubations. 16. Incubate each well for 1 hr at RT with 20 µl of purified cytokine-specific Reagents required antibody or appropriate immunoglobulin isotype control diluted in 1. Fixation Buffer: 5% formalin (10% formalin, CMS, Cat. No. 245-684) PharMingen’s Cytokine IHC Diluent Buffer supplemented with saponin. is dissolved in phosphate buffered-saline (PBS) (Bacto® FA Buffer, 17. Wash slides 2X in PBS with 5 min incubations. Difco Laboratories, Cat. No. 2314-15-0). 18. Incubate each well with 20 µl of a biotinylated secondary antibody diluted 2. Endogenous Peroxidase Blocking Buffer: DAKO Peroxidase Blocking in IHC Cytokine Diluent Buffer for 30 min at RT. Reagent (DAKO, Cat. No. S2001). 19. Wash 2X in PBS with 5 min incubations. 3. Endogenous Biotin Blocking Buffer: Biotin/Avidin Blocking Kit (Vector Laboratories, Cat. No. SP-2001). 20. Apply 20 µl of Vectastain Elite ABC solution (1:2 diluted) to each well on slides and incubate for 30 min at RT. 4. Antibody dilution buffer: PharMingen’s Cytokine IHC Diluent Buffer supplemented with saponin. 21. Wash slides 2X with PBS with 5 minutes incubations. 5. Microscopic slides: Adhesion Slides (Erie Scientific Company, Cat. No. 22. Incubate with 3-3´-Diaminobenzidine tetra hydrochloride (DAB), (Vector, ER-202B-AD) or for cytospins, Colorfrost®/Plus slides (Fisher, Cat. No. Cat. No. SK4100) for less than 5 min at RT. 12-550-17). 23. Stop the development of the color reaction by washing with PBS. 6. Detection system: Vectastain® Elite ABC kit (Vector, Cat. No. PK-6100). 24. The slides are subsequently mounted in short-term storage mounding medium. 7. Mounting medium for short-term storage: Aqua-mount® (Lerner Laboratories, Cat. No.13800). Cytospins 1. Assemble the Cytospin’s sample chamber (e.g. Cytospin 3, Shandon, UK or Secondary antibodies comparable centrifuge), filter card, slide and cytospin racks according to 1. Biotin goat anti-rat IgG (PharMingen, Cat. No. 20392D) manufacturer’s specifications. 6 2. Biotin goat anti-mouse IgG (PharMingen, please inquire) 2. Load 40 µl of approximately 1 x 10 cells to each sample chamber. 3. Spin slides at 600 rpm for 2 min. Procedure for immunocytochemical staining of single-cell preparations 4. Take slides out of the cytospin rack and place them on a staining rack. This procedure describes the immunoenzymatic technique of staining cytokines 5. For fixation and staining please follow the steps 4 through 24 specified within individual cells that are immobilized on microscopic slides via adherence above for staining cells on adhesion slides. (adherent slides) or centrifugation (cytospins).

Note: PharMingen is currently developing an ICC Staining Kit that will include fixation Adhesion slides buffer, antibody diluent buffer, endogenous peroxidase blocking buffer and detection 1. Harvest cells and wash them twice in PBS using centrifugation (400 x g for system (please inquire). 5 min) to remove residual protein. 2. Adjust the cell concentration at 4 x 106 to 5 x 106 cells/ml in PBS. 3. Place 20 µl of the cell suspension in each well of the adhesion slides and let them adhere at room temperature (RT) for 20 min. Please note that the slides should be washed in PBS at RT for 5 min before transferring the cells. 4. Fix cells on slides using fixation buffer for 15 min at RT. 5. Wash slides 2X in PBS with 5 min incubations. 6. Block slides with PBS supplemented with 1% (w/v) BSA (Sigma, Cat. No. A43-78) for 30 min at RT or 10 min at 37°C. 7. Wash slides 2X in PBS and proceed with staining or air dry them and store them at -80˚C for future use. 8. Incubate slides with 20 µl of 1% goat serum and PBS with 0.1% (w/v) saponin for 30 min at RT. 9. Wash slides 2X with PBS with 5 min incubations. 10. Block endogenous peroxidase activity with Endogenous Peroxidase Blocking Buffer (20 µl/well) for 10 min at RT. 11. Wash 2X in PBS with 5 min incubations. 12. Incubate each well with Avidin (20 µl/well) for 15 min. 13. Wash 2X in PBS with 5 min incubations. 14. Incubate each well with Biotin (20 µl/well) for 15 min.

Orders 1-800-848-MABS (6227)• Phone 619-812-8800 Technical Assistance 1-800-TALK-TEC (825-5832) • Fax 619-812-8888 12XXWorld Wide Web http://www.pharmingen.com World Wide Web http://www.pharmingen.com13XX Cytokine Immunocytochemistry Protocol 15. Wash 2X in PBS with 5 min incubations. 16. Incubate each well for 1 hr at RT with 20 µl of purified cytokine-specific Reagents required antibody or appropriate immunoglobulin isotype control diluted in 1. Fixation Buffer: 5% formalin (10% formalin, CMS, Cat. No. 245-684) PharMingen’s Cytokine IHC Diluent Buffer supplemented with saponin. is dissolved in phosphate buffered-saline (PBS) (Bacto® FA Buffer, 17. Wash slides 2X in PBS with 5 min incubations. Difco Laboratories, Cat. No. 2314-15-0). 18. Incubate each well with 20 µl of a biotinylated secondary antibody diluted 2. Endogenous Peroxidase Blocking Buffer: DAKO Peroxidase Blocking in IHC Cytokine Diluent Buffer for 30 min at RT. Reagent (DAKO, Cat. No. S2001). 19. Wash 2X in PBS with 5 min incubations. 3. Endogenous Biotin Blocking Buffer: Biotin/Avidin Blocking Kit (Vector Laboratories, Cat. No. SP-2001). 20. Apply 20 µl of Vectastain Elite ABC solution (1:2 diluted) to each well on slides and incubate for 30 min at RT. 4. Antibody dilution buffer: PharMingen’s Cytokine IHC Diluent Buffer supplemented with saponin. 21. Wash slides 2X with PBS with 5 minutes incubations. 5. Microscopic slides: Adhesion Slides (Erie Scientific Company, Cat. No. 22. Incubate with 3-3´-Diaminobenzidine tetra hydrochloride (DAB), (Vector, ER-202B-AD) or for cytospins, Colorfrost®/Plus slides (Fisher, Cat. No. Cat. No. SK4100) for less than 5 min at RT. 12-550-17). 23. Stop the development of the color reaction by washing with PBS. 6. Detection system: Vectastain® Elite ABC kit (Vector, Cat. No. PK-6100). 24. The slides are subsequently mounted in short-term storage mounding medium. 7. Mounting medium for short-term storage: Aqua-mount® (Lerner Laboratories, Cat. No.13800). Cytospins 1. Assemble the Cytospin’s sample chamber (e.g. Cytospin 3, Shandon, UK or Secondary antibodies comparable centrifuge), filter card, slide and cytospin racks according to 1. Biotin goat anti-rat IgG (PharMingen, Cat. No. 20392D) manufacturer’s specifications. 6 2. Biotin goat anti-mouse IgG (PharMingen, please inquire) 2. Load 40 µl of approximately 1 x 10 cells to each sample chamber. 3. Spin slides at 600 rpm for 2 min. Procedure for immunocytochemical staining of single-cell preparations 4. Take slides out of the cytospin rack and place them on a staining rack. This procedure describes the immunoenzymatic technique of staining cytokines 5. For fixation and staining please follow the steps 4 through 24 specified within individual cells that are immobilized on microscopic slides via adherence above for staining cells on adhesion slides. (adherent slides) or centrifugation (cytospins).

Mouse Positive Controls Specificity Clone Isotype Size Cat . No. Price ($) Various in vitro methods have been reported for stimulating cytokine- IL-2 JES6-1A12 rat IgG 0.25 mg 20191C 250 2a producing cells. These methods include polyclonal activators such as phorbol IL-3 MP2-8F8 rat IgG1 0.25 mg 20201C 250 IL-4 11B11 rat IgG1 0.25 mg 20211C 250 esters plus calcium ionophore, phytohemaglutinin, Staphylococcus enterotoxin IL-10 JES5-16E3 rat IgG2b 0.25 mg 20221C 250 B, or monoclonal antibodies directed against subunits of the TCR/CD3 α TNF- MP6-XT22 rat IgG1 0.25 mg 20231C 250 complex (with or without antibodies directed against costimulatory receptors, γ IFN- XMG1.2 rat IgG1 0.25 mg 20241C 250 such as, CD28). For details on cell stimulation please refer to PharMingen’s GM-CSF MP1-22E9 rat IgG 0.25 mg 20251C 250 2a Cytokine/Chemokine Applications Manual.

Human Negative controls Specificity Clone Isotype Size Cat . No. Price ($) To assure specificity of staining one or more of the following negative controls IL-2 MQ1-17H12 rat IgG2a 0.25 mg 20281C 250 are recommended: IL-5 JES1-39D10 rat IgG 0.25 mg 20371C 250 2a • Immunoglobulin isotype controls: Stain cells using an antibody with the same IL-6 MQ2-6A3 rat IgG2a 0.25 mg 20291C 250 IL-10 JES3-19F1 rat IgG2a 0.25 mg 20381C 250 isotype but with unrelated specificity as that of the cytokine-specific antibody. IL-13 JES10-5A2 rat IgG1 0.25 mg 20301C 250 GM-CSF BVD2-21C11 rat IgG2a 0.25 mg 20311C 250 • Ligand blocking control: Pre-block anti-cytokine antibodies with purified α TNF- MAB11 mouse IgG1 0.25 mg 20321C 250 recombinant cytokine protein for 1 hr at RT. Note: Not all preparations of Isotype controls recombinant cytokine protein are useful for this control (e.g., mouse and human IFN-γ). The binding of antibody-cytokine complexes to cytokine Isotype Clone Size Cat . No. Price ($) receptors may be a factor in the poor blocking capacity of some recombinant Rat IgG R3-34 0.25 mg 20331C 250 1 cytokine proteins. Rat IgG2a R35-95 0.25 mg 20341C 250 Rat IgG2b R35-38 0.25 mg 20361C 250 Mouse IgG1 A112.2 0.25 mg 20351C 250 • Negative control: Stain cells that are not actively producing cytokines. For example, non-activated cells can be used as a negative control. Secondary Antibodies Specificity Size Cat . No. Price ($) Biotin Polyclonal Goat anti-rat IgG 0.5mg 20392D 250

Cytokine Ligand Blocking Controls

Mouse Recombinant protein Size Cat. No. Price ($) L-2 10 µg 19211T 250 IL-3 10 µg 19221T 210 IL-4 5 µg 19231V 275 IL-10 5 µg 19281V 295 TNF-α 10 µg 19321T 250 GM-CSF 10 µg 19291T 395

Human Recombinant protein Size Cat. No. Price ($) IL-2 10 µg 19621T 195 IL-5 5 µg 19651V 275 IL-6 5 µg 19661V 185 IL-10 5 µg 19701V 250 IL-13 5 µg 19731V 250 GM-CSF 5 µg 19741V 375 TNF-α 10 µg 19761T 195

Cytokine Ligand Blocking Controls

Mouse Recombinant protein Size Cat. No. Price ($) L-2 10 µg 19211T 250 IL-3 10 µg 19221T 210 IL-4 5 µg 19231V 275 IL-10 5 µg 19281V 295 TNF-α 10 µg 19321T 250 GM-CSF 10 µg 19291T 395

Orders 1-800-848-MABS (6227)• Phone 619-812-8800 Technical Assistance 1-800-TALK-TEC (825-5832) • Fax 619-812-8888 14World Wide Web http://www.pharmingen.com World Wide Web http://www.pharmingen.com15 PharMingen’s Cytokine Immunocytochemistry and Immunohistochemistry Program PharMingen’s Cytokine Immunocytochemistry Program is a major new facet of our continuing effort to develop reagents and techniques that allow for the successful study of cytokine and chemokine networks at the cellular and molecular level. PharMingen scientists are currently investigating the application of Immunohistochemical methods and reagents that can be used to detect cytokine-producing cells within tissue sections. The information that one obtains by studying cytokine expression in situ in tissue sections is unique because histological information is retained unlike techniques that analyze cells in suspension. Immunohistochemical staining is a particularly powerful technique for examining cytokine-driven responses that can be localized within specific anatomical compartments of the body. For example, it is now appreciated that in certain autoimmune, inflammatory or infectious diseases (e.g., rheumatoid arthritis and HIV infection) the representations of cytokine- producing cells at the local sites of autoimmune or inflammatory responses can be very different from those found in peripheral blood samples from the same individual (Smeets et al., 1998, Andersson et al., 1998, Barnes et al., 1993, Sparrelid et al., 1997, Raqib et al., 1995). Thus, the ability to detect cells producing a particular pattern of cytokines in situ should provide important new insights into the mechanisms of the cytokine network in health and disease.

Currently Available: References • Technical Data Sheets for all PharMingen products for easy downloading 1. Andersson, J., J. Abrams, L. Björk, K. Funa, M. Litton, K. Agren, and U. Andersson. 1994. Concomitant in vivo production of 19 different cytokines in human tonsils. Immunol. 83:16-24. • Complete catalog search of PharMingen’s 3,000 products 2. Andersson, J., L. Björk, C. A. Dinarello, H. Towbin, and U. Andersson. 1992. Lipopolysaccharide induces human interleukin- 1 receptor antagonist and interleukin-1 production in the same cell. Eur J Immunol 22:2617-2623. 3. Andersson, J.,T. E. Fehniger, B. K. Patterson, J. Pottage, M. Angoli, P. Jones, H. Behbahani and A. Landay. 1998. Early • Hot New Product Announcements reduction of immune activation in lymphoid tissue following highly active HIV therapy. AIDS 12:F123-F129. 4. Barnes, P.F., S. Lu, J. S. Abrams, E. Wang, M. Yamamura, R. L. Modlin. 1993. Cytokine production at the site of disease in human tuberculosis. Infect Immun 61(8):3482-3489. • Special offers for free promotional items, posters, etc. 5. Björk, L. 1995. The characteristics of the intracellular cytokine pattern. In Development of an image analysis system for the evaluation of cytokine production. Thesis, Stockholm. 6. Björk, L., T. E. Fehniger, U. Andersson, and J. Andersson. 1996. Computerized assessment of production of multiple human • Online registration to join our catalog and electronic mailing lists cytokines at the single-cell level using image analysis. J Leukoc Biol 59:287-295. 7. Gordon, M. Y. 1991. Hemopoietic growth factors and receptors: bound and free. Cancer Cells 3:127-133. 8. Hsu, S. M., L. Raine, and H. Fanger. 1981a. Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase Upcoming Features for 1999: techniques: a comparison between ABC and unlabeled antibody (PAP) procedures. J Histochem Cytochem 29:577-580. 9. Hsu, S. M., L. Raine, and H. Fanger. 1981b. A comparative study of the peroxidase-antiperoxidase method and an avidin- • Expanded product information sections biotin complex method for studying polypeptide hormones with radioimmunoassay antibodies. Am J Clin Pathol 75:734-738. 10. Raqib, R., A. A. Lindberg, L. Bjork, P. K. Bardhan, B. Wretlind, U. Andersson, and J. Andersson. 1995. Down-regulation of gamma interferon, tumor necrosis factor type I, interleukin 1 (IL-1) type I, IL-3, IL-4, and transforming growth factor beta type • Online ordering I receptors at the local site during the acute phase of Shigella infection. Infect Immun 63:3079. 11. Sander, B., J. Andersson, and U. Andersson. 1991. Assessment of cytokines by immunofluorescence and the paraformaldehyde-saponin procedure. Immunol. Rev. 119:65-93. • Customer education sections focusing on hot areas of research 12. Smeets,T. J., R. J. E. M Dolhain, A. M. Miltenburg, R. de Kuiper, F. C. Breedveld, P. P. Tak. 1998. Poor expression of T cell- derived cytokines and activation and proliferation markers in early rheumatoid synovial tissue. Clin Immunol Immunopathol (including Apoptosis, Cytokines/Chemokines, etc.) 88(1):84-90. 13. Sparrelid, E., D. Emanuel, T. Fehniger, U. Andersson, and J. Andersson. 1997. Interstitial pneumonitis in bone marrow transplant recipients is associated with local production of TH2-type cytokines and lack of T cell-mediated cytotoxicity. Transplantation 63:1782. 14. Stevenson, F. T., F. Torrano, R. M. Locksley, and D. H. Lovett. 1992. Interleukin 1: the patterns of translation and intracellular distribution support alternative secretory mechanisms. J Cell Physiol 152:223-231. 15. Suni, M. A., L. J. Picker, and V. C. Maino. 1998. Detection of antigen-specific T cell cytokine expression in whole blood by flow cytometry. J Immunol Methods 212:89-98. 16. Walter, P., R. Gilmore, and G. Blobel. 1984. Protein translocation across the endoplasmic reticulum. Cell 38:5-8. 17. Willingham, M.C. and I. Pastan. 1985. An atlas of immunofluorescence in cultured cells. Academic Press, Inc. London, p 5.

Orders 1-800-848-MABS (6227)• Phone 619-812-8800 16World Wide Web http://www.pharmingen.com PharMingen’s Cytokine Immunocytochemistry and Immunohistochemistry Program PharMingen’s Cytokine Immunocytochemistry Program is a major new facet of our continuing effort to develop reagents and techniques that allow for the successful study of cytokine and chemokine networks at the cellular and molecular level. PharMingen scientists are currently investigating the application of Immunohistochemical methods and reagents that can be used to detect cytokine-producing cells within tissue sections. The information that one obtains by studying cytokine expression in situ in tissue sections is unique because histological information is retained unlike techniques that analyze cells in suspension. Immunohistochemical staining is a particularly powerful technique for examining cytokine-driven responses that can be localized within specific anatomical compartments of the body. For example, it is now appreciated that in certain autoimmune, inflammatory or infectious diseases (e.g., rheumatoid arthritis and HIV infection) the representations of cytokine- producing cells at the local sites of autoimmune or inflammatory responses can be very different from those found in peripheral blood samples from the same individual (Smeets et al., 1998, Andersson et al., 1998, Barnes et al., 1993, Sparrelid et al., 1997, Raqib et al., 1995). Thus, the ability to detect cells producing a particular pattern of cytokines in situ should provide important new insights into the mechanisms of the cytokine network in health and disease.

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