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
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).
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).
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.
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.