CD34 (ICO115) Mouse mAb #3569

Immunohistochemistry (Paraffin)

A. Solutions and Reagents

1. Xylene
2. Ethanol, anhydrous denatured, histological grade (100% and 95%)
3. Deionized water (dH₂O)
4. Hematoxylin (optional)
5. Wash Buffer:
   1. 1X Tris Buffered Saline with Tween^® 20 (TBST): To prepare 1L 1X TBST add 100 ml 10X Tris Buffered Saline with Tween^® 20 (#9997) to 900 ml dH₂0, mix.
   2. Antibody Diluent TBST/5% Normal Goat Serum: to 5 mL 1X TBST, add 250 µl Normal Goat Serum #5425).
6. 1X Citrate Unmasking Solution: To prepare 250 mL of 1X citrate unmasking solution, dilute 25 ml of SignalStain^® Citrate Unmasking Solution (10X) (#14746) with 225 mL of dH₂O.
7. 3% Hydrogen Peroxide: To prepare, add 10 ml 30% H₂O₂ to 90 ml dH₂O.
8. Blocking Solution: TBST/5% Normal Goat Serum or 1X Animal-Free Blocking Solution.
   1. TBST/5% Normal Goat Serum: to 5 ml 1X TBST, add 250 µl Normal Goat Serum (#5425).
   2. 1X Animal-Free Blocking Solution: to 4 mL of dH₂O add 1 ml of Animal-Free Blocking Solution (5X) (#15019).
9. Detection System: VECTASTAIN^® Elite ABC, including biotinylated secondary antibody (Vector Laboratories).
10. Substrate: Vector^® NovaRED™ (Vector Laboratories).
11. Hematoxylin: Hematoxylin (#14166).
12. Mounting Medium: SignalStain^® Mounting Medium (#14177).

B. Deparaffinization/Rehydration

NOTE: Do not allow slides to dry at any time during this procedure.

1. Deparaffinize/hydrate sections:
   1. Incubate sections in three washes of xylene for 5 minutes each.
   2. Incubate sections in two washes of 100% ethanol for 10 minutes each.
   3. Incubate sections in two washes of 95% ethanol for 10 minutes each.
2. Wash sections twice in dH₂O for 5 minutes each.

C. Antigen Unmasking

For Citrate: Heat slides in a microwave submersed in 1X citrate unmasking solution until boiling is initiated; follow with 10 min at a sub-boiling temperature (95°-98°C). Cool slides on bench top for 30 min.

D. Staining

1. Wash sections in dH₂O three times for 5 minutes each.
2. Incubate sections in 3% hydrogen peroxide for 10 minutes.
3. Wash sections in dH₂O twice for 5 minutes each.
4. Wash sections in wash buffer for 5 minutes.
5. Block each section with 100-400 µl of preferred blocking solution for 1 hour at room temperature.
6. Remove blocking solution and add 100-400 µl primary antibody diluted in recommended antibody diluent to each section. Incubate overnight at 4°C.
7. Prepare ABC solution per manufacturer's recommendations.
8. Remove primary antibody and wash section three times with wash buffer for 5 minutes each.
9. Add 100-400 µl biotinylated secondary antibody, diluted in TBST per manufacturer’s recommendation, to each section. Incubate 30 minutes at room temperature.
10. Remove secondary antibody solution and wash sections three times with wash buffer for 5 minutes each.
11. Cover sections with 100-400 µl pre-mixed ABC solution as needed and incubate in a humidified chamber for 30 min at room temperature.
12. Wash section three times with wash buffer for 5 min each.
13. Prepare Vector^® NovaRED™ per manufacturer's recommendations.
14. Apply 100-400 µl substrate to each section and monitor closely. 1-10 minutes generally provides an acceptable staining intensity.
15. If desired, counterstain sections with hematoxylin (#14166).
16. Wash sections in dH₂O two times for 5 minutes each.
17. Dehydrate sections:
    1. Incubate sections in 95% ethanol two times for 10 seconds each.
    2. Repeat in 100% ethanol, incubating sections two times for 10 seconds each.
    3. Repeat in xylene, incubating sections two times for 10 seconds each.
18. Mount sections with coverslips and mounting medium (#14177).

Flow Cytometry, Live Cell Protocol for Unconjugated Mouse Antibodies

A. Solutions and Reagents

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

1. 1X Phosphate Buffered Saline (PBS): To prepare 1 L 1X PBS: add 100 ml 10X PBS (#12528) to 900 ml water mix.
2. Antibody Dilution Buffer: Purchase ready-to-use Flow Cytometry Antibody Dilution Buffer (#13616), or prepare a 0.5% BSA PBS buffer by dissolving 0.5 g Bovine Serum Albumin (BSA) (#9998) in 100 ml 1X PBS. Store at 4°C.
3. 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) #59997

NOTE: When including fluorescent cellular dyes in your experiment (including viability dyes, DNA dyes, etc.), please refer to the dye product page for the recommended protocol. Visit www.cellsignal.com for a full listing of cellular dyes validated for use in flow cytometry.

B. Immunostaining

NOTE: Count cells using a hemocytometer or alternative method.

NOTE: If using whole blood, lyse red blood cells and wash by centrifugation prior to Immunostaining.

NOTE: Optimal centrifugation conditions will vary depending upon cell type and reagent volume. Generally, 150-300g for 1-5 minutes will be sufficient to pellet the cells.

1. Aliquot desired number of cells into tubes or wells. (Generally, 5x10⁵ to 1x10⁶ cells per assay.)
2. Pellet cells by centrifugation and remove supernatant.
3. Resuspend cells in 100 µl of diluted primary antibody, prepared in Antibody Dilution Buffer at a recommended dilution or as determined via titration.
4. Incubate for 30 min to 1 hr on ice. Protect from light.
5. Wash by centrifugation in Antibody Dilution Buffer. Discard supernatant. Repeat.
6. Resuspend cells in 100 µl of diluted fluorochrome-conjugated secondary antibody (prepared in Antibody Dilution Buffer at the recommended dilution).
7. Incubate for 30 min on ice. Protect from light.
8. Wash by centrifugation in Antibody Dilution Buffer. Discard supernatant. Repeat.
9. Resuspend cells in 200-500 µl of Antibody Dilution Buffer and analyze on flow cytometer.