Ki-67 (8D5) Mouse mAb #9449

Immunofluorescence (Immunocytochemistry)

A. Solutions and Reagents

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.

1. 20X Phosphate Buffered Saline (PBS): (9808) To prepare 1L 1X PBS: add 50 ml 20X PBS to 950 ml dH₂O, mix. Adjust pH to 8.0.
2. Formaldehyde: 16%, methanol free, Polysciences, Inc. (cat# 18814), use fresh and store opened vials at 4°C in dark, dilute in 1X PBS for use.
3. Blocking Buffer: (1X PBS / 5% normal serum / 0.3% Triton™ X-100): To prepare 10 ml, add 0.5 ml normal serum from the same species as the secondary antibody (e.g., Normal Goat Serum (#5425) to 9 ml 1X PBS) and mix well. While stirring, add 30 µl Triton™ X-100.
4. Antibody Dilution Buffer: (1X PBS / 1% BSA / 0.3% Triton™ X-100): To prepare 10 ml, add 30 µl Triton™ X-100 to 10 ml 1X PBS. Mix well then add 0.1 g BSA (#9998), mix.

5. Recommended Fluorochrome-conjugated Anti-Mouse secondary antibodies:

   • Anti-Mouse IgG (H+L), F(ab')₂ Fragment (Alexa Fluor^® 488 Conjugate) #4408
   • Anti-Mouse IgG (H+L), F(ab')₂ Fragment (Alexa Fluor^® 555 Conjugate) #4409
   • Anti-Mouse IgG (H+L), F(ab')₂ Fragment (Alexa Fluor^® 594 Conjugate) #8890
   • Anti-Mouse IgG (H+L), F(ab')₂ Fragment (Alexa Fluor^® 647 Conjugate) #4410
6. Prolong^® Gold AntiFade Reagent (#9071), Prolong^® Gold AntiFade Reagent with DAPI (#8961).

B. Specimen Preparation - Cultured Cell Lines (IF-IC)

NOTE: Cells should be grown, treated, fixed and stained directly in multi-well plates, chamber slides or on coverslips.

1. Aspirate liquid, then cover cells to a depth of 2–3 mm with 4% formaldehyde diluted in 1X PBS.

   NOTE: Formaldehyde is toxic, use only in a fume hood.

2. Allow cells to fix for 15 min at room temperature.
3. Aspirate fixative, rinse three times in 1X PBS for 5 min each.
4. Proceed with Immunostaining (Section C).

C. Immunostaining

NOTE: All subsequent incubations should be carried out at room temperature unless otherwise noted in a humid light-tight box or covered dish/plate to prevent drying and fluorochrome fading.

1. Block specimen in Blocking Buffer for 60 min.
2. While blocking, prepare primary antibody by diluting as indicated on datasheet in Antibody Dilution Buffer.
3. Aspirate blocking solution, apply diluted primary antibody.
4. Incubate overnight at 4°C.
5. Rinse three times in 1X PBS for 5 min each.
6. Incubate specimen in fluorochrome-conjugated secondary antibody diluted in Antibody Dilution Buffer for 1–2 hr at room temperature in the dark.
7. Rinse three times in 1X PBS for 5 min each.
8. Coverslip slides with Prolong^® Gold Antifade Reagent (#9071) or Prolong^® Gold Antifade Reagent with DAPI (#8961).
9. For best results, allow mountant to cure overnight at room temperature. For long-term storage, store slides flat at 4°C protected from light.

Flow Cytometry General Protocol

If using whole blood, please follow the Flow Cytometry Whole Blood Protocol.

A. Solutions and Reagents

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.

1. 20X Phosphate Buffered Saline (PBS): (#9808) To prepare 1 L 1X PBS: add 50 ml 20X PBS to 950 ml dH2O, mix.
2. 16% Formaldehyde (methanol free).
3. 100% methanol.
4. Incubation Buffer: Dissolve 0.5 g Bovine Serum Albumin (BSA) (#9998) in 100 ml 1X PBS. Store at 4°C.
5. Recommended Anti-Mouse secondary antibodies::
   • Anti-Mouse IgG (H+L), F(ab')₂ Fragment (Alexa Fluor^® 488 Conjugate) #4408
   • Anti-Mouse IgG (H+L), F(ab')₂ Fragment (Alexa Fluor^® 594 Conjugate) #8890
   • Anti-Mouse IgG (H+L), F(ab')₂ Fragment (Alexa Fluor^® 647 Conjugate) #4410
   • Anti-Mouse IgG (H+L), F(ab')₂ Fragment (PE Conjugate) #8887

B. Fixation

1. Collect cells by centrifugation and aspirate supernatant.
2. Resuspend cells in 0.5–1 ml 1X PBS. Add formaldehyde to obtain a final concentration of 4%.
3. Fix for 10 min at 37°C.
4. Chill tubes on ice for 1 min.
5. For extracellular staining with antibodies that do not require permeabilization, proceed to immunostaining (Section D) or store cells in PBS with 0.1% sodium azide at 4°C; for intracellular staining, proceed to permeabilization (Section C).

C. Permeabilization

NOTE: This step is critical for many CST antibodies.

1. Permeabilize cells by adding ice-cold 100% methanol slowly to pre-chilled cells, while gently vortexing, to a final concentration of 90% methanol. Alternatively, remove fix prior to permeabilization by centrifugation and resuspend in 90% methanol as described above.
2. Incubate 30 min on ice.
3. Proceed with immunostaining (Section D) or store cells at -20°C in 90% methanol.

D. Immunostaining

NOTE: Account for isotype matched controls for monoclonal antibodies or species matched IgG for polyclonal antibodies. Count cells using a hemocytometer or alternative method.

1. Aliquot 0.5–1 x 10⁶ cells into each assay tube (by volume).
2. Add 2–3 ml incubation buffer to each tube and wash by centrifugation. Repeat.
3. Resuspend cells in 100 μl of diluted primary antibody (prepared in incubation buffer at the recommended dilution).
4. Incubate for 1 hr at room temperature.
5. Wash by centrifugation in 2–3 ml incubation buffer.
6. Resuspend cells in fluorochrome-conjugated secondary antibody, diluted in incubation buffer at the recommended dilution.
7. Incubate for 30 min at room temperature.
8. Wash by centrifugation in 2–3 ml incubation buffer.
9. Resuspend cells in 0.5 ml PBS and analyze on flow cytometer; alternatively, for DNA staining, proceed to optional DNA stain (Section E).

E. Optional DNA Dye

1. Resuspend cells in 0.5 ml of DNA dye (e.g. Propidium Iodide (PI)/RNase Staining Solution #4087).
2. Incubate for at least 30 min at room temperature.
3. Analyze cells in DNA staining solution on flow cytometer.

Flow Cytometry Whole Blood Protocol

If using cell lines, please follow the Flow Cytometry General Protocol.

A. Solutions and Reagents

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.

1. 20X Phosphate Buffered Saline (PBS): (#9808) To prepare 1 L 1X PBS: add 50 ml 20X PBS to 950 ml dH₂O, mix.
2. 16% Formaldehyde (methanol free).
3. Triton™ X-100: To prepare 50 ml of 0.1% Triton™ X-100 add 50 μl Triton™ X-100 to 50 ml 1 X PBS and mix well.
4. 50% methanol.
5. Incubation Buffer: Dissolve 0.5 g Bovine Serum Albumin (BSA) (#9998) in 100 ml 1X PBS. Store at 4°C.
6. Secondary Antibodies:
   • Anti-Mouse IgG (H+L), F(ab')₂ Fragment (Alexa Fluor^® 488 Conjugate) #4408
   • Anti-Mouse IgG (H+L), F(ab')₂ Fragment (Alexa Fluor^® 594 Conjugate) #8890
   • Anti-Mouse IgG (H+L), F(ab')₂ Fragment (Alexa Fluor^® 647 Conjugate) #4410

B. Preparation of Whole Blood (fixation, lysis, and permeabilization) for Immunostaining

1. Aliquot 100 μl fresh whole blood per assay tube.
2. OPTIONAL: Place tubes in rack in 37°C water bath for short-term treatments with ligands, inhibitors, drugs, etc.
3. Add 65 μl of 10% formaldehyde to each tube.
4. Vortex briefly and let stand for 15 min at room temperature.
5. Add 1 ml of 0.1% Triton™ X-100 to each tube.
6. Vortex and let stand for 30 min at room temperature.
7. Add 1 ml incubation buffer.
8. Pellet cells by centrifugation and aspirate supernatant.
9. Repeat steps 7 and 8.
10. Resuspend cells in ice-cold 50% methanol in PBS (store methanol solution at -20°C until use).
11. Incubate at least 10 min on ice.
12. Proceed with staining or store cells at -20°C in 50% methanol.

C. Staining Using Unlabeled Primary and Conjugated Secondary Antibodies

NOTE: Account for isotype-matched controls for monoclonal antibodies or species matched IgG for polyclonal antibodies.

1. Add 2–3 ml incubation buffer to each tube and rinse by centrifugation. Repeat.
2. Add primary antibodies diluted as recommended on datasheet or product webpage in incubation buffer.
3. Incubate for 30–60 min at room temperature.
4. Wash by centrifugation in 2–3 ml incubation buffer.
5. Resuspend cells in fluorochrome-conjugated secondary antibody diluted in incubation buffer according to the manufacturer’s recommendations.
6. Incubate for 30 min at room temperature.
7. Wash by centrifugation in 2–3 ml incubation buffer.
8. Resuspend cells in 0.5 ml PBS and analyze on flow cytometer.

Reference: Chow S, Hedley D, Grom P, Magari R, Jacobberger JW, Shankey TV (2005) Whole blood fixation and permeabilization protocol with red blood cell lysis for flow cytometry of intracellular phosphorylated epitopes in leukocyte subpopulations. Cytometry A 67(1), 4–17.