references
 
 
 

Calbindin D28k

Calbindin D-28k is a vitamin D-dependent calcium-binding protein. In normal animals, the CaBP protein appeared from Postnatal Day 3 in the granule cells of the DG and from Day 5 in the CA1-CA2 pyramidal cells of Ammon's horn. The cells of both regions showed positive CaBP labelling about 1 week after their formation. The sequence of labelling of the granule cells was a reflection of the major sequences of neurogenesis. Also CaBP could not be detected in hippocampal cells until dendritic arborization and axon growth had occurred (Rami et al., 1987). This protein is useful in determination of state of maturation of granule cells in the DG of the hippocampus.
   
 

Calbindin D28k Protocol

Positive Control Tissue: Rat hippocampus fixed with 4% paraformaldehyde.

Tissue: Rat Hippocampus

Fixation: 4% Paraformaldehyde in PBS pH 7.2

Primary Antibody: Rabbit anti-Calbindin D28K

Ideal Dilution Ratio 1:500-1000

Secondary Antibody: Alexa 488/568 Goat anti-rabbit IgG (H+L), (Molecular Probes, Cat# ).

Optimal dilution 1:200

  • Sections were washed with PBS to remove any sodium azide.
  • Calbindin labelling: sections were incubated in primary antibody to Calbindin (1:500 in 0.3% Triton-X solution, Anti-CaBP antibody made in rabbit, Chemicon) for 72 hours at 4 C.
  • Wash in PBS for 3x5min.
  • The resulting sections were incubated for 2 hours in the dark at room temperature in fluorescent-conjugated secondary antibody (Alexa 488/568, goat anti-rabbit, Chemicon).
  • Wash in PBS for 3x5min.
  • Wash in distilled water 2x5min.
  • Coverslip with mounting medium (Permafluor for fluorecent tissues and Permount for DAB tissues).