Calretinin Antibody Staining Protocol for Immunohistochemistry


Description: Calretinin is an intracellular calcium-binding protein belonging to the troponin C superfamily characterised by a structural motif described as the EF-hand domain. Calcium is an important moderator of a number of vital physiological processes, including neuronal excitability, axonal transport, synthesis and release of some neurotransmitters, membrane permeability and enzyme activity. Calretinin possesses six characteristic EF-hand structures of which five are able to bind calcium ions with high affinity. Calretinin is found in the nervous system and thymus. Calretinin can also be demonstrated at precise moments of the cell cycle, in particular, G1 phase and mitosis, and is associated with kinetochore microtubules which may indicate a role in chromosome separation. The immunohistochemical detection of calretinin in developing cerebellum has been reported to be restricted to the later stages indicated by weak staining from week 21 of gestation, in Purkinje and basket cells and in neurons of the dentate nucleus. This intensity of staining increases as the cerebellum matures. Other related calcium-binding proteins such as calbindin 28kD and parvalbumin have been reported to be expressed at earlier stages of development.


Primary Antibody

Name: Calretinin Antibody

Clone: 5A5, Mouse anti-Human

Supplier: Novocastra Labs

Catalog Number: NCL-Calretinin

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: 60min/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: Predominantly cytoplasmic but nuclear staining may also be observed
Images: Search image

Additional Information:
Tissue Type: Testis, cerebellum, thymus
Fixation: Formalin fixed paraffin sections
Positive Control: Testis, cerebellum, thymus
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