Upstream / Downstream
Explore pathways related to this product.
CST Antibody Performance Guarantee
To Purchase # 12202S
|12202T||20 µl (80 sections)||$99.00.0|
|12202S||100 µl (400 sections)||$246.00.0|
Find answers on our FAQs page.
- Additional protein information
- Analytical tools
Ki-67 (D3B5) Rabbit mAb (Mouse Preferred; IHC Formulated) #12202
Immunohistochemical analysis of paraffin-embedded mouse colon using Ki-67 (D3B5) Rabbit mAb (Mouse Preferred; IHC Formulated).Learn more about how we get our images
Immunohistochemical analysis of paraffin-embedded mouse heart using Ki-67 (D3B5) Rabbit mAb (Mouse Preferred; IHC Formulated). Heart is negative, as expected.Learn more about how we get our images
Gallery: Ki-67 (D3B5) Rabbit mAb (Mouse Preferred; IHC Formulated) #12202
A. Solutions and Reagents
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
- Ethanol, anhydrous denatured, histological grade (100% and 95%).
- Deionized water (dH2O).
- Hematoxylin (optional).
- Wash Buffer:
- 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.
- SignalStain® Antibody Diluent (#8112).
- 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.
- 3% Hydrogen Peroxide: To prepare 100 ml, add 10 ml 30% H2O2 to 90 ml dH2O.
- Blocking Solution: TBST/5% Normal Goat Serum or 1X Animal-Free Blocking Solution.
- Detection System: SignalStain® Boost IHC Detection Reagents (HRP, Rabbit #8114).
- Substrate: SignalStain® DAB Substrate Kit (#8059).
- Hematoxylin: Hematoxylin (#14166).
- Mounting Medium: SignalStain® Mounting Medium (#14177).
NOTE: Do not allow slides to dry at any time during this procedure.
- Deparaffinize/hydrate sections:
- Incubate sections in three washes of xylene for 5 min each.
- Incubate sections in two washes of 100% ethanol for 10 min each.
- Incubate sections in two washes of 95% ethanol for 10 min each.
- Wash sections two times in dH2O for 5 min 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.
- Wash sections in dH2O three times for 5 min each.
- Incubate sections in 3% hydrogen peroxide for 10 min.
- Wash sections in dH2O two times for 5 min each.
- Wash sections in wash buffer for 5 min.
- Block each section with 100–400 µl of preferred blocking solution for 1 hr at room temperature.
- Remove blocking solution and add 100–400 µl primary antibody diluted in SignalStain® Antibody Diluent (#8112) to each section. Incubate overnight at 4°C.
- Equilibrate SignalStain® Boost Detection Reagent (HRP, Rabbit #8114) to room temperature.
- Remove antibody solution and wash sections with wash buffer three times for 5 min each.
- Cover section with 1–3 drops SignalStain® Boost Detection Reagent (HRP, Rabbit #8114) as needed. Incubate in a humidified chamber for 30 min at room temperature.
- Wash sections three times with wash buffer for 5 min each.
- Add 1 drop (30 µl) SignalStain® DAB Chromogen Concentrate to 1 ml SignalStain® DAB Diluent and mix well before use.
- Apply 100–400 µl SignalStain® DAB to each section and monitor closely. 1–10 min generally provides an acceptable staining intensity.
- Immerse slides in dH2O.
- If desired, counterstain sections with hematoxylin (#14166).
- Wash sections in dH2O two times for 5 min each.
- Dehydrate sections:
- Incubate sections in 95% ethanol two times for 10 sec each.
- Repeat in 100% ethanol, incubating sections two times for 10 sec each.
- Repeat in xylene, incubating sections two times for 10 sec each.
- Mount sections with coverslips and mounting medium (#14177).
posted February 2010
revised March 2016
Ki-67 (D3B5) Rabbit mAb (Mouse Preferred; IHC Formulated) recognizes endogenous levels of murine Ki-67 protein. It will also detect endogenous levels of human Ki-67 protein; however, Ki-67 (D2H10) Rabbit mAb (IHC Specific) #9027 is recommended for the detection of human Ki-67 protein in paraffin-embedded tissues.Species Reactivity: Mouse
Monoclonal antibody is produced by immunizing animals with a recombinant protein specific to the amino terminus of Ki-67 protein.
Ki-67, named after the location where it was discovered (Kiel University, Germany), is a nuclear nonhistone protein (1) that is universally expressed among proliferating cells and absent in quiescent cells (2). Ki-67 detects proliferating cells in G1, S, G2, and mitosis, but not in the G0 resting phase. Research studies have shown that high levels of Ki-67 are associated with poorer breast cancer survival (3). Research studies have explored the use of Ki-67, along with other markers, as potential prognostic or predictive markers in breast cancer and other malignant diseases (4).
For Research Use Only. Not For Use In Diagnostic Procedures. Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.