Multi-Cytokeration Antibody Staining Protocol for Immunohistochemistry

 

Description: Cytokeratins are a family of more than 20 structurally related proteins that are differentially expressed in different epithelia and at various stages of differentiation and development. Cytokeratins belong to two families, type I (acidic) and type II (basic), which are usually co-ordinately synthesized in pairs so that at least one member of each family is expressed in each epithelial cell. NCL-C11 reacts with simple epithelium and both basal and suprabasal layers of cornifying and non-cornifying squamous epithelium.

 

Primary Antibody

Name: Multi-Cytokeratin (4/5/6/8/10/13/18) Antibody

Clone:  C-11, Mouse anti-Human

Supplier: Novocastra Labs

Catalog Number: NCL-C11

Dilution: 1:20 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 minutes/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:
Tissue Type: Skin
Fixation: Formalin fixed paraffin sections, or acetone fixed frozen sections.
Positive Control: Skin
Negative Control: Omit primary antibody, isotype control or 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

Notes:

There is no need to perform antigen retrieval for frozen sections.

 

References: