ER alpha Antibody Staining Protocol for Immunohistochemistry

 

Description: Estrogen receptors (ER) are members of the steroid/thyroid hormone receptor superfamily of ligand-activated transcription factors. Estrogen receptors, including ERα and ERβ, contain DNA binding and ligand binding domains and are critically involved in regulating the normal function of reproductive tissues. ERα and ERβ have been shown to be differentially activated by various ligands. Receptor-ligand interaction trigger a cascade of events, including dissociation from heat shock proteins, receptor dimerization, phosphorylation and the association of the hormone activated receptor with specific regulatory elements in target genes. Evidence suggests that ERα and ERβ may be regulated by distinct mechanisms even though they share many functional characteristics.

 

Primary Antibody

Name: Estrogen Receptor (ER) alpha Antibody

Clone: Rabbit anti-Mouse

Supplier: Santa Cruz Biotechnology

Catalog Number: sc-542

Dilution: 1:300 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 ºC - 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: Nuclear
Images: Search image

Additional Information:
Tissue Type: Breast carcinoma, uterus.
Fixation: Formalin fixed paraffin sections.
Positive Control: Breast carcinoma, uterus.
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:

Not tested on frozen sections

 

References: