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.

Begin with transferring the sections from the antifreeze solution to TBS with the help of a fine brush. Rinse the sections thoroughly, starting with an overnight wash at 4 degrees Celsius, followed by five 10-minute washes at room temperature. All these incubations should be performed with continuous gentle agitation.

If one of the antigens of interest is a thymidine analog, a hydrochloric acid denaturation step has to be implemented at this point, followed by a neutralization step in borate buffer. Proceed with permeabilization and blocking of the unspecific antibody-binding sites in TBS-plus. Let this incubation go for 1 hour at room temperature.

Then, incubate in the first primary antibody diluted in TBS-plus overnight at 4 degrees Celsius. The next day, wash the sections three times in TBS, and once in TBS-T. Let each bath go for 10 minutes. Then, incubate in first fluorochrome-conjugated secondary antibody for three hours at room temperature. From now on, protect the sections from light exposure.

After another wash series, transfer the sections into a 10% host species serum solution from which the two primary antibodies were made. Incubate the sections in this solution for three hours to saturate the open paratopes on the first secondary antibody. Later, rinse three times in TBS, and once in TBS-T to remove the serum.

Then, prepare a solution of TBS-plus with 50 micrograms per milliliter of unconjugated monovalent Fab fragments directed against the host species of the primary antibody. This covers the epitopes that could otherwise be recognized by the second secondary antibody. Transfer the sections into this solution and incubate overnight at 4 degrees Celsius.

After the incubation, rinse the sections at least three times in TBS, and once in TBS-T. During transfers, swab the mesh inserts containing the sections on a paper towel to remove residual Fab leavings. Now, incubate the sections in the second primary antibody overnight at 4 degrees Celsius.

After another wash series, apply the second secondary antibody for three hours at room temperature. Finally, wash the sections three times in TBS and proceed with mounting the sections in gelatin. Air-dry the slides and coverslip them using an anti-fade mounting medium.