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Purified Mouse Anti-Human MCP-1
Purified Mouse Anti-Human MCP-1

Immunocytochemistry analysis of MCP-1 expression on human peripheral blood mononuclear cells. PBMC were isolated from human peripheral blood by density gradient centrifugation and were cultured overnight at 37°C with LPS (Sigma No. L-8274, 1 µg/ml) in the presence of GolgiStop™ (Cat. No. 554724). The activated cells were harvested and the level of MCP-1 producing cells was detected by immunocytochemistry using a three-step staining procedure that employs a Biotin Goat anti-mouse IgG secondary antibody (Cat. No. 550337) Streptavidin-HRP (Cat. No. 550946) and DAB Substrate Kit (Cat. No. 550880).

Immunocytochemistry analysis of MCP-1 expression on human peripheral blood mononuclear cells. PBMC were isolated from human peripheral blood by density gradient centrifugation and were cultured overnight at 37°C with LPS (Sigma No. L-8274, 1 µg/ml) in the presence of GolgiStop™ (Cat. No. 554724). The activated cells were harvested and the level of MCP-1 producing cells was detected by immunocytochemistry using a three-step staining procedure that employs a Biotin Goat anti-mouse IgG secondary antibody (Cat. No. 550337) Streptavidin-HRP (Cat. No. 550946) and DAB Substrate Kit (Cat. No. 550880).

Product Details
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BD Pharmingen™
CCL2; C-C motif chemokine 2; Chemokine (C-C motif) ligand 2; MCAF; SCYA2
Human (QC Testing), Rhesus, Cynomolgus (Tested in Development)
Mouse IgG1, κ
Recombinant Human MCP-1
ELISA (Routinely Tested)
0.5 mg/ml
AB_2071656
Aqueous buffered solution containing ≤0.09% sodium azide.
RUO


Preparation And Storage

The monoclonal antibody was purified from tissue culture supernatant or ascites by affinity chromatography. Store undiluted at 4°C.

Recommended Assay Procedures

Immunocytochemistry: The purified format of the 5D3-F7 antibody (Cat. No. 550416) can be used to identify and enumerate human MCP-1 producing cells by immunocytochemistry. For optimal indirect immunocytochemical staining, the 5D3-F7 antibody should be titrated (≤ 1 µg) and visualized via a three-step staining procedure in combination with Biotin Goat Anti-Mouse IgG (Cat. No. 550337) Streptavidin-HRP (Cat. No. 550946) and a DAB detection reagent (Cat. No. 550880). Please see protocol for a detailed description of the immunocytochemical procedure.

CYTOKINE IMMUNOCYTOCHEMISTRY PROTOCOL

REAGENTS REQUIRED

1. Fixation Buffer: 5% formalin (10% formalin, CMS, Cat. No. 245-684) is dissolved in phosphate buffered-saline (PBS) (Bacto  FA Buffer, Difco Laboratories, Cat. No.  2314-15-0), or BD Pharmingen™ ICC Fixation Buffer (BD Cat. No. 550010)

2. Endogenous Peroxidase Blocking Buffer: DAKO Peroxidase Blocking Reagent (DAKO, Cat. No. S2001).

3. Endogenous Biotin Blocking Buffer: Biotin/Avidin Blocking Kit (Vector Laboratories, Cat. No. SP-2001).

4. Antibody dilution buffer: BD™ Pharmingen Antibody Diluent for IHC (Cat. No. 559148) supplemented with saponin.

5. Microscopic slides: Adhesion Slides (Erie Scientific Company, Cat. No. ER-202B-AD) or for cytospins, Colorfrost /Plus slides (Fisher, Cat. No. 12-550-17).

6. Detection system: BD Pharmingen Streptavidin-horseradish peroxidase (HRP), (Cat. No. 550946) or the Anti-Mouse Ig HRP Detection Kit (Cat. No. 551011).

7. Mounting medium for short-term storage: Aqua-mount® (Lerner Laboratories, Cat. No. 13800).

8. DAB Substrate Kit (contains 3-3 -Diaminobenzidine tetra hydrochloride), (BD Cat. No. 550880) or Anti-Mouse Ig HRP Detection Kit (Cat. No. 551011).

SECONDARY ANTIBODIES

1. Biotin Goat anti-Mouse IgG (Cat. No. 550337) or the Anti-Mouse Ig HRP Detection Kit (Cat. No. 551011).

PROCEDURE FOR IMMUNOCYTOCHEMICAL STAINING OF SINGLE-CELL PREPARATIONS

This procedure describes the immunoenzymatic technique of staining cytokines within individual cells that are immobilized on microscopic slides via adherence (adherent slides) or centrifugation (cytospins).  

ADHESION SLIDES

1. Harvest cells and wash them twice in PBS using centrifugation (400 x g for 5 min) to remove residual protein.

2. Adjust the cell concentration at 4-5 x 10e6 cells/ml in PBS.

3. Wash slides for 5 minutes in PBS at room temperature (RT) before transferring cells. Place 20 µl of the cell suspension in each well of the adhesion slides and let them adhere at RT for 20 min.

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) 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.

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 antibody or appropriate immunoglobulin isotype control diluted in Pharmingen's Antibody Diluent Buffer for IHC supplemented with saponin.

17. Wash slides 2X in PBS with 5 min incubations.

18. Incubate each well with 20 µl of a biotinylated secondary antibody diluted in Antibody Diluent Buffer for IHC Buffer for 30 min at RT.

19. Wash 2X in PBS with 5 min incubations.

20. Apply 20 µl of Streptavidin-HRP (BD Cat. No. 550946)  to each well on slides and incubate for 30 min at RT.

21. Wash slides 2X with PBS with 5 minutes incubations.

22. Incubate with DAB Substrate as directed, (BD Cat. No. 550880) for less than 5 min at RT.

23. Stop the development of the color reaction by washing with PBS.

24. The slides are subsequently mounted in short-term storage mounding medium.

CYTOSPINS

1. Assemble the Cytospin's sample chamber (e.g. Cytospin 3, Shandon, UK or comparable centrifuge), filter card, slide and cytospin racks according to manufacturer's specifications.

2. Load 40 µl of approximately 1 x 10e6 cells to each sample chamber.

3. Spin slides at 600 rpm for 2 min.

4. Take slides out of the cytospin rack and place them on a staining rack.

5. For fixation and staining please follow the steps 4 through 24 specified above for staining cells on adhesion slides.

Product Notices

  1. Since applications vary, each investigator should titrate the reagent to obtain optimal results.
  2. Caution: Sodium azide yields highly toxic hydrazoic acid under acidic conditions. Dilute azide compounds in running water before discarding to avoid accumulation of potentially explosive deposits in plumbing.
  3. Sodium azide is a reversible inhibitor of oxidative metabolism; therefore, antibody preparations containing this preservative agent must not be used in cell cultures nor injected into animals. Sodium azide may be removed by washing stained cells or plate-bound antibody or dialyzing soluble antibody in sodium azide-free buffer. Since endotoxin may also affect the results of functional studies, we recommend the NA/LE (No Azide/Low Endotoxin) antibody format, if available, for in vitro and in vivo use.
  4. Species cross-reactivity detected in product development may not have been confirmed on every format and/or application.
  5. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
550416 Rev. 3
Antibody Details
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5D3-F7

The 5D3-F7 monoclonal antibody specifically binds to human monocyte chemoattractant protein-1 (MCP-1), also known as CCL2 (C-C motif chemokine 2), Monocyte chemotactic and activating factor (MCAF), and Small-inducible cytokine A2 (SCYA2). MCP-1 is a 10-14 kDa glycoprotein member of the beta or CC family of chemokines. It expressed by monocytes, fibroblasts, endothelial cells and other cell types in response to IL-1, IL-6, TNF, and a variety of other stimuli. MCP-1 binds to and exerts its biological activity through G-protein coupled chemokine receptors including CCR2/CD192 and CCR4/CD194. It serves as a chemoattractant and activating factor for monocytes and other cell types including T cells, NK cells, and basophils.

MCP-1 is a member of the CC chemokine family and it is produced by monocytes, T lymphocytes, fibroblasts, endothelial cells, smooth muscle cells, keratinocytes and some tumors. Its production can be induced by LPS or IFN-γ. Clone 5D3-F7 also cross reacts with an intracellular component of LPS-stimulated (24 hours) peripheral blood monocytes of rhesus and cynomolgus macaque monkeys. The staining pattern observed on non-human primate monocytes is not as strong as that seen on normal human peripheral blood monocytes.

550416 Rev. 3
Format Details
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Purified
Tissue culture supernatant is purified by either protein A/G or affinity purification methods. Both methods yield antibody in solution that is free of most other soluble proteins, lipids, etc. This format provides pure antibody that is suitable for a number of downstream applications including: secondary labeling for flow cytometry or microscopy, ELISA, Western blot, etc.
Purified
550416 Rev.3
Citations & References
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Development References (5)

  1. Hsu SM, Raine L, Fanger H. A comparative study of the peroxidase-antiperoxidase method and an avidin-biotin complex method for studying polypeptide hormones with radioimmunoassay antibodies. Am J Clin Pathol. 1981; 75(5):734-738. (Biology). View Reference
  2. Hsu SM, Raine L, Fanger H. Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabeled antibody (PAP) procedures. J Histochem Cytochem. 1981; 29(4):577-580. (Biology). View Reference
  3. Peri G, Milanese C, Matteucci C, et al. A new monoclonal antibody (5D3-F7) which recognizes human monocyte-chemotactic protein-1 but not related chemokines. Development of a sandwich ELISA and in situ detection of producing cells. J Immunol Methods. 1994; 174(1-2):249-257. (Biology). View Reference
  4. Rollins BJ, Stier P, Ernst T, Wong GG. The human homolog of the JE gene encodes a monocyte secretory protein. Mol Cell Biol. 1989; 9(11):4687-4695. (Biology). View Reference
  5. Yoshimura T, Yuhki N, Moore SK, Appella E, Lerman MI, Leonard EJ. Human monocyte chemoattractant protein-1 (MCP-1). Full-length cDNA cloning, expression in mitogen-stimulated blood mononuclear leukocytes, and sequence similarity to mouse competence gene JE. FEBS Lett. 1989; 244(2):487-493. (Biology). View Reference
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550416 Rev. 3

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Comparisons, where applicable, are made against older BD Technology, manual methods or are general performance claims.  Comparisons are not made against non-BD technologies, unless otherwise noted.

For Research Use Only. Not for use in diagnostic or therapeutic procedures.