Immunofluorescence Staining for Visualizing Proliferated Neural Stem Cells in Mouse Brain Sections

Take fixed mouse brain sections containing neural cells, including proliferated neural stem cells labeled with EdU, a thymidine analog.

Outline the sections with a hydrophobic marking to prevent solution spread.

Add a detergent to permeabilize the tissue. Wash to remove excess detergent.

Add a fluorophore to label the EdU. Remove the excess fluorophores.

Using a fluorescence microscope, confirm proper staining of the EdU-labeled cells.

Apply a blocking buffer containing proteins to prevent non-specific antibody binding. Remove the excess proteins.

Incubate with primary antibodies that bind to specific proteins in neural stem cells. Remove the unbound antibodies.

Add fluorophore-conjugated secondary antibodies to bind the primary antibodies. Wash off the unbound antibodies.

Stain the nuclei with a dye and remove the excess dye.

Remove the hydrophobic marking, apply mounting media, and place a coverslip.

Using a confocal microscope, observe the sections to visualize the labeled neural stem cells.

To stain the tissue sections with thymidine analog, remove the tissue sections from the citrate buffer. Let the tissue sections dry and completely adhere to the slides before drawing a border with a hydrophobic pen. Next, permeabilize the sections with permeabilization buffer for 20 to 30 minutes. Wash the sections twice using TBS Triton for 5 minutes each time. Then, prepare an EdU reaction solution.

Incubate the sections in EdU reaction solution for 30 minutes to an hour. Subsequently, wash them three times in TBS Triton for 5 minutes each time. At this stage, check if the EdU reaction works by using a fluorescent microscope. EdU-labeled cells should be observed if the reaction works. Now, block the mounted tissue sections using blocking buffer raised in the same animal as the secondary antibody for 30 minutes to an hour. Then, wash them twice in TBS Triton for 5 minutes each time.

During the blocking step, prepare primary antibody solution by mixing the primary antibody in blocking buffer. Subsequently, add 250 microliters of primary antibody solution per slide to ensure the tissue sections are completely submerged, and incubate them overnight at room temperature. The next day, wash the tissue sections three times in TBS Triton for 5 minutes each to remove excess primary antibody.

Then, incubate the tissue sections in fluorophore-conjugated secondary antibody prepared in blocking buffer solution for two hours at room temperature. Wash the tissue sections three times in TBS Triton for 5 minutes each to remove excess secondary antibody. Next, apply 300 micromolar DAPI solution at 1-to-100 in PBS for 15 minutes at room temperature. Afterward, wash the tissue sections three times in PBS for five minutes each to remove excess DAPI, and remove the PAP pen circle around the tissue using a cotton swab. Let the sections dry before applying mounting media and covering them with coverslips.