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PhosphoSitePlus® Resource

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REACTIVITY SENSITIVITY MW (kDa) Isotype
H Endogenous Rabbit IgG
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Immunohistochemistry (Paraffin)

Immunohistochemical analysis of paraffin-embedded human ovarian clear cell adenocarcinoma using OX40L (D6K7R) Rabbit mAb (IHC Specific). Analysis was performed on the Leica® Bond™ Rx.

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Immunohistochemistry (Paraffin)

Immunohistochemical analysis of paraffin-embedded HUVEC cell pellet (left, positive) or MCF7 cell pellet (right, negative) using OX40L (D6K7R) Rabbit mAb (IHC Specific).

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Immunohistochemistry (Paraffin)

Immunohistochemical analysis of paraffin-embedded human colon carcinoma using OX40L (D6K7R) Rabbit mAb (IHC Specific).

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Immunohistochemistry (Paraffin)

Immunohistochemical analysis of paraffin-embedded human prostate carcinoma using OX40L (D6K7R) Rabbit mAb (IHC Specific).

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Immunohistochemistry (Paraffin)

Immunohistochemical analysis of paraffin-embedded human tonsil using OX40L (D6K7R) Rabbit mAb (IHC Specific).

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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

Product Usage Information

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

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.

Specificity / Sensitivity

OX40L (D6K7R) Rabbit mAb recognizes endogenous levels of total OX40L protein. Staining of unknown specificity has been observed in skeletal muscle.


Species Reactivity: Human

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Ala15 of human OX40L protein.

OX40 (TNFRSF4, CD134) is a member of the tumor necrosis factor (TNF) receptor superfamily that regulates T cell activity and immune responses. The OX40 protein contains four cysteine rich domains, a transmembrane domain, and a cytoplasmic tail containing a QEE motif (1,2). OX40 is primarily expressed on activated CD4+ and CD8+ T-cells, while the OX40 ligand (OX40L, TNFSF4, CD252) is predominantly expressed on activated antigen presenting cells (3-7). The engagement of OX40 with OX40L leads to the recruitment of TNF receptor-associated factors (TRAFs) and results in the formation of a TCR-independent signaling complex. One component of this complex, PKCθ, activates the NF-κB pathway (2,8). OX40 signaling through Akt can also enhance TCR signaling directly (9). Research studies indicate that the OX40L-OX40 pathway is associated with inflammation and autoimmune diseases (10). Additional research studies show that OX40 agonists augment anti-tumor immunity in several cancer types (11,12).


1.  Croft, M. (2010) Annu Rev Immunol 28, 57-78.

2.  So, T. and Croft, M. (2012) Front Immunol 3, 133.

3.  Paterson, D.J. et al. (1987) Mol Immunol 24, 1281-90.

4.  Mallett, S. et al. (1990) EMBO J 9, 1063-8.

5.  Dürkop, H. et al. (1995) Br J Haematol 91, 927-31.

6.  Godfrey, W.R. et al. (1994) J Exp Med 180, 757-62.

7.  Al-Shamkhani, A. et al. (1997) J Biol Chem 272, 5275-82.

8.  So, T. et al. (2011) Proc Natl Acad Sci U S A 108, 2903-8.

9.  So, T. and Croft, M. (2013) Front Immunol 4, 139.

10.  Gough, M.J. and Weinberg, A.D. (2009) Adv Exp Med Biol 647, 94-107.

11.  Weinberg, A.D. et al. (2011) Immunol Rev 244, 218-31.

12.  Linch, S.N. et al. (2015) Front Oncol 5, 34.


Entrez-Gene Id 7292
Swiss-Prot Acc. P23510


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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OX40L (D6K7R) Rabbit mAb (IHC Specific)