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24326
CD16 (D1N9L) Rabbit mAb

CD16 (D1N9L) Rabbit mAb #24326

APPLICATIONS

REACTIVITY SENSITIVITY MW (kDa) Isotype
H Endogenous Rabbit IgG
IHC-Leica® Bond™

Immunohistochemical analysis of paraffin-embedded human ovarian serous carcinoma using CD16 (D1N9L) Rabbit mAb performed on the Leica® BOND™ Rx.

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IHC-P (paraffin)

Immunohistochemical analysis of paraffin-embedded purified CD56+ human peripheral blood mononuclear cell pellet (left, positive) or Raji cell pellet (right, negative) using CD16 (D1N9L) Rabbit mAb.

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IHC-P (paraffin)

Immunohistochemical analysis of paraffin-embedded human colon carcinoma using CD16 (D1N9L) Rabbit mAb.

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IHC-P (paraffin)

Immunohistochemical analysis of paraffin-embedded human lung adenocarcinoma using CD16 (D1N9L) Rabbit mAb.

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IHC-P (paraffin)

Immunohistochemical analysis of paraffin-embedded human non-Hodgkin's lymphoma using CD16 (D1N9L) Rabbit mAb.

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Immunohistochemistry (Leica® BOND)

NOTE: Please see product datasheet or product webpage for appropriate antibody dilution.

  Step Reagents Time/Temperature

1.

Dewax

BOND Dewax Solution, 100% Alcohol, BOND Wash Solution

Pre-programmed Leica®BOND

2.

Antigen Retrieval

BOND Epitope Retrieval ER2 Solution

20 min., 100°C

3.

Peroxide Blocking

Peroxide Block*

5 min.

 

WASH

BOND Wash Solution

3x 0:00 min.

4.

Protein Block

CST #5425 NGS or #15019 Animal-Free Blocking Solution

20 min.

5.

Primary Antibody

Dilute in #8112 Antibody Diluent

30 min.

 

WASH

BOND Wash Solution

3x 2:00 min.

6.

Secondary Detection

Novolink Polymer*

10 min.

 

WASH

BOND Wash Solution/Deionized Water

Custom (see below)

7a.

Visualization

Mixed DAB Refine*

0:00 min.

7b.

Visualization

Mixed DAB Refine*

10 min.

 

WASH

Deionized Water

3x 0:00 min.

8.

Counterstain

Hematoxylin*

5 min.

 

WASH

Deionized Water

3x 0:00 min.

 

WASH

BOND Wash Solution

3x 0:00 min.

 

WASH

Deionized Water

3x 0:00 min.

9.

Dehydration (Offline):
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).

  Custom wash: BOND Wash Solution 2:00
    BOND Wash Solution Dispenser Type: OPEN 0:00
    BOND Wash Solution 2:00
    BOND Wash Solution Dispenser Type: OPEN 0:00
    BOND Wash Solution 0:00
    Deionized Water 0:00

*Reagent included in BOND Polymer Refine Detection Kit
Catalog No: DS9800

LEICA® is a registered trademark of Leica®Microsystems IR GmbH.

BOND is a trademark of Leica®Biosystems Melbourne Pty. Ltd.

No affiliation or sponsorship between CST and Leica®Microsystems IR GmbH or Leica®Biosystems Melbourne Pty. Ltd. is implied.

posted February 2017

Protocol Id: 1444

Immunohistochemistry (Paraffin)

A. Solutions and Reagents

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

  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.
  6. SignalStain® Antibody Diluent (#8112).
  7. 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.
  8. 3% Hydrogen Peroxide: To prepare 100 ml, add 10 ml 30% H2O2 to 90 ml dH2O.
  9. 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).
  10. Detection System: SignalStain® Boost IHC Detection Reagents (HRP, Rabbit #8114).
  11. Substrate: SignalStain® DAB Substrate Kit (#8059).
  12. Hematoxylin: Hematoxylin (#14166).
  13. 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 min each.
    2. Incubate sections in two washes of 100% ethanol for 10 min each.
    3. Incubate sections in two washes of 95% ethanol for 10 min each.
  2. 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.

D. Staining

  1. Wash sections in dH2O three times for 5 min each.
  2. Incubate sections in 3% hydrogen peroxide for 10 min.
  3. Wash sections in dH2O two times for 5 min each.
  4. Wash sections in wash buffer for 5 min.
  5. Block each section with 100–400 µl of preferred blocking solution for 1 hr at room temperature.
  6. Remove blocking solution and add 100–400 µl primary antibody diluted in SignalStain® Antibody Diluent (#8112) to each section. Incubate overnight at 4°C.
  7. Equilibrate SignalStain® Boost Detection Reagent (HRP, Rabbit #8114) to room temperature.
  8. Remove antibody solution and wash sections with wash buffer three times for 5 min each.
  9. 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.
  10. Wash sections three times with wash buffer for 5 min each.
  11. Add 1 drop (30 µl) SignalStain® DAB Chromogen Concentrate to 1 ml SignalStain® DAB Diluent and mix well before use.
  12. Apply 100–400 µl SignalStain® DAB to each section and monitor closely. 1–10 min generally provides an acceptable staining intensity.
  13. Immerse slides in dH2O.
  14. If desired, counterstain sections with hematoxylin (#14166).
  15. Wash sections in dH2O two times for 5 min each.
  16. Dehydrate sections:
    1. Incubate sections in 95% ethanol two times for 10 sec each.
    2. Repeat in 100% ethanol, incubating sections two times for 10 sec each.
    3. Repeat in xylene, incubating sections two times for 10 sec each.
  17. Mount sections with coverslips and mounting medium (#14177).

posted February 2010

revised March 2016

Protocol Id: 283

Application Dilutions
IHC-Leica® Bond™ 1:200
Immunohistochemistry (Paraffin) 1:400
Storage:

Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.

CD16 (D1N9L) Rabbit mAb recognizes endogenous levels of total CD16 protein. This antibody detects CD16A and CD16B.

Species Reactivity:

Human

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Pro32 of human CD16 protein.

CD64 (FcgammaRI), CD32 (FcgammaRII) and CD16 (FcgammaRIII) are three classes of the immunoglobulin superfamily. CD64 has a high affinity for IgG with three Ig-like domains while CD32 and CD16 have low affinities with two Ig-like domains. Two genes encode CD16-A and CD16-B resulting only in a 6 amino acid difference in their ectodomains. However, CD16-A has a transmembrane anchor versus CD16-B, which has a glycosylphosphatidylinositol (1). CD64, CD32 and CD16 are membrane glycoproteins that are expressed by all immunologically active cells and trigger various immune functions (activate B cells, phagocytosis, antibody-dependent cellular cytotoxicity, immune complex clearance and enhancement of antigen presentation) (2). CD16 cross-linking induces tyrosine phosphorylation (Tyr394) of Lck in NK cells (3). CD32 has tyrosine-based activation motifs in the cytoplasmic domain in contrast to CD16, which associates with molecules possessing these motifs (1).

CD16A is expressed by NK cells, macrophages, and a subset of monocytes, while CD16B is expressed by neutrophils (4). CD16 is commonly used in combination with CD56 to characterize NK cells, with CD16 identifying NK cells capable of cytotoxicity (5).

  1. Maenaka, K. et al. (2001) J. Biol. Chem. 276, 44898-44904.
  2. Fridman, W. H. et al. (1992) Immunol. Rev. 125, 49-76.
  3. Pignata, C. et al. (1993) J. Immunol. 151, 6794-6800.
  4. Pincetic, A. et al. (2014) Nat Immunol 15, 707-16.
  5. Nagler, A. et al. (1989) J Immunol 143, 3183-91.
Entrez-Gene Id
2214
Swiss-Prot Acc.
P08637
For Research Use Only. Not For Use In Diagnostic Procedures.

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
SignalStain is a trademark of Cell Signaling Technology, Inc.
BOND is a trademark of Leica Biosystems Melbourne Pty. Ltd. No affiliation or sponsorship between CST and Leica Microsystems IR GmbH or Leica Biosystems Melbourne Pty. Ltd is implied.
LEICA is a registered trade​mark of Leica Microsystems IR GmbH.

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