Insulin A Antibody Staining Protocol for Immunohistochemistry


Description: Insulin is a secreted peptide hormone that elicits metabolic effects such as increases in glucose uptake and glycogen synthesis leading to a decrease in blood glucose concentration. Insulin is first formed as a precursor molecule, preproinsulin, which is later cleaved to proinsulin and finally to the mature insulin hormone. Mature insulin consists of 51 amino acids, contained within an A chain and a B chain that are connected by two disulfide bridges. It increases cell permeability to monosaccharides, amino acids and fatty acids. Insulin is secreted by the pancreas at basal levels in the absence of exogenous stimuli, with secretion increasing in response to glucose. Insulin action is effected by the binding of insulin to cell-surface receptors on the target cell membrane. Defects of insulin are the cause of hyperproinsulinemia and of type II diabetes mellitus.


Primary Antibody

Name: Insulin A (C-12) Antibody

Clone: Goat polyclonal

Supplier: Santa Cruz Biotechnology

Catalog Number: sc-7839

Dilution: 1:100 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

Reagent: Mayer's Hematoxylin
Staining Time: 30 seconds

Staining Pattern: Membrane/cytoplasmic
Images: Search image

Additional Information:
Species Reactivity: Human, mouse, rat
Fixation: Formalin fixed paraffin sections
Positive Control: Pancreas
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