Phospho-p44/42 Map Kinase Antibody Staining Protocol for Immunohistochemistry

 

Description: Both p44 and p42 MAP kinases (Erk1 and Erk2) function in a protein kinase cascade that plays a critical role in the regulation of cell growth and differentiation. MAP kinases are activated by a wide variety of extracellular signals including growth and neurotrophic factors, cytokines, hormones and neurotransmitters. Activation of MAP kinases occurs through
phosphorylation of threonine and tyrosine (202 and 204 of human MAP kinase [Erk1] or 183 and 185 of rat Erk2) at the sequence T*EY* by a single upstream MAP kinase kinase (MEK) (5,6). Both kinases are known to weakly autophosphorylate on tyrosine.

 

Primary Antibody

Name: Phospho-p44/42 Map Kinase (Thr202/Tyr204) Antibody

Clone: Rabbit polyclonal

Supplier: Cell Signaling Technology

Catalog Number: 9101

Dilution: 1:200 using IHC-TekTM Antibody Diluent (Cat# IW-1000 or IW-1001) to reduce background and unspecific staining and serum blocking step is NOT needed.

Incubation Time/Temp: 60 min/room temperature


Antigen Retrieval
Device: IHC-TekTM Epitope Retrieval Steamer Set (Cat# IW-1102)
Buffer/pH value: IHC-TekTM Epitope Retrieval Solution (Cat# IW-1100)
Heat/Cool Temperature: 95-100 ºC/room temperature
Heat/Cool Time: 20 minutes/20 minutes

Detection Methods
Standard Method: ABC Method or LSAB Method
Enhanced Method: Polymeric Methods

Chromogen Substrate
Reagent: DAB
Incubation Time/Temperature: 1-3 minutes/room temperature

Counterstain
Reagent: Mayer's Hematoxylin
Staining Time: 30 seconds

Results:
Staining Pattern: Cytoplasmic
Images: Search image

Additional Information:
Species Reactivity: Human, mouse, rat, hamster, chicken, zebra fish
Fixation: Formalin fixed paraffin sections, or acetone fixed frozen sections (no antigen retrieval is needed)
Positive Control: Ischemia brain
Negative Control: Omit primary antibody, isotype control, absorption control
Blocking: 2-5% normal serum to reduce unspecific background staining; 0.5-3% H2O2 to block endogenous peroxidase activity; avidin/biotin to block endogenous biotin activity if necessary

 

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