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Immunohistochemical analysis of paraffin-embedded E14 mouse embryo using Cleaved Caspase-3 (Asp175) (D3E9) Rabbit mAb #9579 (left) or Rabbit (DA1E) mAb IgG XP® SignalStain® Isotype Control #12960 (right).

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Immunohistochemical analysis of paraffin-embedded Kelly xenograft using Cleaved Caspase-3 (Asp175) (D3E9) Rabbit mAb #9579 (left) or Rabbit (DA1E) mAb IgG XP® SignalStain® Isotype Control #12960 (right).

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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.

    NOTE: Additional 10X TBST will be required for washes.

    1. Tris Buffered Saline with Tween® 20 (TBST-10X) #9997: To prepare wash buffer add 100 μl of Tris Buffered Saline with Tween® 20 (TBST-10X) #9997 to 900 ml of dH2O. OR
    2. 10X Tris Buffered Saline (TBS): To prepare 1 L, add 24.2 g Trizma® base (C4H11NO3) and 80 g sodium chloride (NaCl) to 1 L dH2O. Adjust pH to 7.6 with concentrated HCl. 1X TBS/0.1% Tween® 20 (1X TBST): To prepare 1 L, add 100 ml 10X TBS to 900 ml dH2O. Add 1 ml Tween® 20 and mix.
  6. Antibody Diluent: SignalStain® Antibody Diluent #8112
  7. Antigen Unmasking: 10 mM Sodium Citrate Buffer: To prepare 1 L, add 2.94 g sodium citrate trisodium salt dihydrate (C6H5Na3O72H2O) to 1 L dH2O. Adjust pH to 6.0.
  8. 3% Hydrogen Peroxide: To prepare, add 10 ml 30% H2O2 to 90 ml dH2O.
  9. Blocking Solution: 1X TBST/5% normal goat serum: Add 100 μl 10X TBST (#9997) and 50 μl normal goat serum (#5425) to 850 μl dH2O.
  10. Detection System: SignalStain® Boost IHC Detection Reagent (HRP, Rabbit) (#8114).
  11. Substrate: SignalStain® DAB Substrate Kit (#8059), which includes SignalStain® DAB Diluent (#11724) and SignalStain® DAB Chromogen Concentrate (#11725).

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 twice in dH2O for 5 min each.

C. Antigen Unmasking

  1. Bring slides to a boil in 10 mM sodium citrate buffer pH 6.0, then maintain at a sub-boiling temperature for 10 min. 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 twice for 5 min each.
  4. Wash section in wash buffer for 5 min.
  5. Block each section with 100–200 μl blocking solution for 1 hr at room temperature.
  6. Remove blocking solution and add 100–200 μl primary antibody diluted at 1:500 in SignalStain® Antibody Diluent (#8112) to each section. Incubate overnight at 4°C.
    1. Rabbit (DA1E) mAb XP® SignalStain® Isotype Control (#3900) is used as a negative control. Dilute at 1:500 in SignalStain® Antibody Diluent (#8112) and apply 100–200 μl to each section.
  7. Equilibrate SignalStain® Boost Detection Reagent (#8114) to room temperature.
  8. Remove antibody solution and wash sections in wash buffer three times for 5 min each.
  9. Cover section with 1–3 drops SignalStain® Boost Detection Reagent (#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 30 μl SignalStain® DAB Chromogen Concentrate (#11725) to 1 ml SignalStain® DAB Diluent (#11724) and mix well before use.
  12. Apply 100–400 μl SignalStain® DAB to each section and monitor closely. 1–10 minutes generally provides an acceptable staining intensity.
  13. Immerse slides in dH2O.
  14. If desired, counterstain sections in hematoxylin per manufacturer’s instructions.
  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 coverslips.

posted July 2013

protocol id: 64

Product Includes Quantity Isotype
Peroxidase Quench 1 x 15 ml  
Blocking Solution 1 x 15 ml  
Prediluted P-p44/42 MAPK (T202/Y204)(D13.14.4E) XP mAb #4370 1 x 15 ml  
Prediluted Negative Control 1 x 15 ml  
Biotinylated Secondary Ab 1 x 15 ml  
Reagent A 1 x 850 µl  
Reagent B 1 x 850 µl  
NovaRED Substrate 1 (TM) 1 x 600 µl  
NovaRED Substrate 2 (TM) 1 x 600 µl  
NovaRED Substrate 3 (TM) 1 x 600 µl  
NovaRED Substrate 4 (TM) 1 x 800 µl  
Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) Blocking Peptide 1150 1 x 100 µg  
Mixing Bottle x    

Order Details

Custom Ordering Details: The control slides that accompany this kit are cut freshly upon ordering. Please allow three to five days for your order to be processed and shipped.

Product Description

CST's Growth/Proliferation Marker: SignalStain® Phospho-p44/42 MAPK (Thr202/Tyr204) IHC Detection Kit is a "ready to use" system designed to detect the activation of MAP kinase in human tissue and cell preps by immunohistochemistry. The kit utilizes the ABC immunoperoxidase method to detect endogenous levels of phosphorylated p44/42 MAP kinase. Prediluted Phospho-p44/42MAPK (Thr202/Tyr204) (20G11) Rabbit mAb #4376 is bound by a biotinylated secondary antibody. Avidin DH and biotinylated horseradish peroxidase are complexed by mixing in defined amounts prior to use, and the mixture subsequently binds the secondary antibody. The macromolecular complex is localized by incubation with NovaRED™ enzyme substrate.

The prediluted primary antibody, along with the ABC system, allows the user consistently to examine phosphorylated-p44/42 MAP Kinase localization and offers the highest sensitivity with the lowest background.


Specificity / Sensitivity

Growth/Proliferation Marker: SignalStain® Phospho-p44/42 MAPK (Thr202/Tyr204) IHC Detection Kit recognizes endogenous levels of p42 and p44 MAP kinase (Erk1 and Erk2) only when phosphorylated at Thr202 and Tyr204 of Erk1 (Thr183 and Tyr185 of Erk2). The antibody does not cross-react with the corresponding phosphorylated residues of either SAPK/JNK or p38 MAP kinase. This kit was developed for and is recommended for immunohistochemistry only.


Species predicted to react based on 100% sequence homology: Mouse, Rat, Monkey, Zebrafish

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Thr202/Tyr204 of human p44 MAP kinase.

Mitogen-activated protein kinases (MAPKs) are a widely conserved family of serine/threonine protein kinases involved in many cellular programs, such as cell proliferation, differentiation, motility, and death. The p44/42 MAPK (Erk1/2) signaling pathway can be activated in response to a diverse range of extracellular stimuli including mitogens, growth factors, and cytokines (1-3), and research investigators consider it an important target in the diagnosis and treatment of cancer (4). Upon stimulation, a sequential three-part protein kinase cascade is initiated, consisting of a MAP kinase kinase kinase (MAPKKK or MAP3K), a MAP kinase kinase (MAPKK or MAP2K), and a MAP kinase (MAPK). Multiple p44/42 MAP3Ks have been identified, including members of the Raf family, as well as Mos and Tpl2/COT. MEK1 and MEK2 are the primary MAPKKs in this pathway (5,6). MEK1 and MEK2 activate p44 and p42 through phosphorylation of activation loop residues Thr202/Tyr204 and Thr185/Tyr187, respectively. Several downstream targets of p44/42 have been identified, including p90RSK (7) and the transcription factor Elk-1 (8,9). p44/42 are negatively regulated by a family of dual-specificity (Thr/Tyr) MAPK phosphatases, known as DUSPs or MKPs (10), along with MEK inhibitors, such as U0126 and PD98059.


1.  Roux, P.P. and Blenis, J. (2004) Microbiol Mol Biol Rev 68, 320-44.

2.  Baccarini, M. (2005) FEBS Lett 579, 3271-7.

3.  Meloche, S. and Pouysségur, J. (2007) Oncogene 26, 3227-39.

4.  Dalby, K.N. et al. (1998) J Biol Chem 273, 1496-505.

5.  Roberts, P.J. and Der, C.J. (2007) Oncogene 26, 3291-310.

6.  Rubinfeld, H. and Seger, R. (2005) Mol Biotechnol 31, 151-74.

7.  Murphy, L.O. and Blenis, J. (2006) Trends Biochem Sci 31, 268-75.

8.  Marais, R. et al. (1993) Cell 73, 381-93.

9.  Kortenjann, M. et al. (1994) Mol Cell Biol 14, 4815-24.

10.  Owens, D.M. and Keyse, S.M. (2007) Oncogene 26, 3203-13.


Entrez-Gene Id 5595, 5594
Swiss-Prot Acc. P27361, P28482


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.
NovaRED™ is a trademark of Vector Laboratories.