An Immunofluorescence Staining Technique to Detect Adult Hippocampal Neurogenesis

Take a murine brain section consisting of the hippocampal dentate gyrus, a site for neurogenesis composed of neuronal progenitors at distinct differentiation stages.

Transfer the section into a permeabilization-blocking solution. Detergent molecules in the solution permeabilize the cells, while the proteins block the non-specific binding sites.

Add a primary antibody cocktail targeting specific neurogenesis markers, a microtubule-associated protein, and an intermediate filament protein.

Remove the unbound antibodies. Introduce different fluorophore-labeled secondary antibodies binding to the primary antibodies.

Add serum proteins from the same host as one of the primary antibodies, saturating the paratopes on the secondary antibodies.

Introduce monovalent Fab fragments that saturate the serum proteins' epitopes, preventing non-specific binding.

Add a second same-host-raised primary antibody, binding to another intermediate filament protein.

Introduce fluorophore-labeled secondary antibodies targeting the second primary antibody.

Using fluorescence microscopy, identify the neuronal progenitor subtypes expressing unique marker combinations.