An Immunofluorescence Staining Technique to Detect Adult Hippocampal Neurogenesis

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Tissue Preparation

1. Prepare 4% formaldehyde in 0.1 M phosphate buffer pH 7.4 on the day before perfusion. Store at 4 °C.
2. Transcardially perfuse the deeply anesthetized mice (3.5% isoflurane) via the left ventricle with 10 ml ice-cold phosphate-buffered saline (PBS), then with 40 ml ice-cold formaldehyde (flow rate 5 ml/min). Dissect the brain and post-fix in the same fixative for 24 hr at 4°C.
3. Transfer the brains consecutively into 10% (24 hr at 4°C) and 30% sucrose (until the brain sinks, approx. 48 hr). For freezing, slowly submerge the cryoprotected brains into -25 °C isopentane until no bubbles emerge from the tissue. Store at -80 °C.
4. Cut coronal sections of 40 µm thickness on a freezing microtome (block temperature at -25 to -16 °C). Sequentially transfer sections into antifreeze solution containing wells of a 24-well cell culture plate (see Figure 1). Store at -20 °C.

2. Immunostaining

NOTE: Sections are processed free-floating, usually in 6-well plates equipped with a carrier plate and mesh inserts. As an exception, blocking, antibody incubations, and avidin-biotin complex (ABC) reactions are done in 12- or 24-well plates without mesh inserts (0.5 to 1 ml per well is sufficient, depending on the number of slices that have to be stained). During these steps, transfer sections with the help of a fine brush (rinse with each new solution). All incubations are done with continuous agitation (max 150 rpm).

1. Immunohistochemistry (ABC method)
   1. Transfer sections from antifreeze into tris-buffered saline (TBS) and rinse thoroughly (once O/N at 4 °C, 5 times at room temperature [RT] for 10 min each) to completely remove antifreeze.
   2. To quench endogenous peroxidase activity, Incubate for 30 min in 1.5% H₂O₂ in TBS-Tween 20 (TBS-T). Pay attention to bubbling and re-submerge sections if necessary. Rinse 3 times in TBS for 15 min each.
   3. Optional: Meanwhile, preheat a heating cabinet and 2 N HCl to 37 °C. To denature DNA, incubate sections for 30 min at 37 °C in 2 N HCl. Gently separate sections with the help of a brush.
   4. Optional: Neutralize sections for 10 min in 0.1 M borate buffer pH 8.5, RT. While transferring, briefly swab the mesh inserts containing the sections on a paper towel to remove HCl leavings. Rinse 2 times in TBS for 15 min each.
   5. Incubate in TBSplus to permeabilize the tissue and block unspecific antibody binding sites for 1 hr at RT.
   6. Incubate in primary antibody diluted in TBSplus, O/N at 4 °C. Rinse 3 times in TBS for 15 min each.
   7. Incubate in biotinylated secondary antibody diluted in TBSplus for 3 hours at RT. Rinse 3 times in TBS for 15 min each. Meanwhile…
   8. Prepare ABC complex according to manufacturer's protocol (1% A + 1% B in TBS-T). Allow to stand for 30 min at RT before use. Incubate sections in AB reagent for 1 hr at RT. Rinse 3 times in TBS for 15 min each.
   9. Prepare 50 ml 0.5 mg/ml 3,3′-Diaminobenzidine (DAB) in TBS-T per 6- or 12-well plate, split into two halves, and pipette 4 ml or 2 ml per well, respectively. Transfer sections into DAB solution (CAUTION! DAB is toxic. Follow the specific MSDS provided by the supplier, i.e., wear a lab coat and gloves and use a chemical fume hood).
   10. Add 0.5 ml 1% H₂O₂ to the remaining 25 ml DAB solution, mix, and pipette equivalent volumes as above to each well to start the peroxidase reaction. Incubate for 12 min. Rinse 3 times in TBS for 15 min each.
   11. Mount sections to slides in gelatin and air dry O/N. Coverslip with permanent mounting medium.
   12. Optional: Counterstain before placing the coverslip.
       NOTE: If the signal-to-noise ratio is low because of the high background, repeat the H₂O₂ treatment after the incubation with AB reagent (step 2.1.8).
2. Multiple-immunofluorescence
   1. Single or simultaneous multiple immunofluorescence
      1. Transfer sections from antifreeze into TBS and rinse thoroughly (once O/N at 4 °C, 5 times at RT for 10 min each) to remove the antifreeze completely.
      2. Optional: as steps 2.1.3 – 2.1.4.
      3. Incubate in TBSplus to permeabilize the tissue and block unspecific antibody binding sites, 1 hr at RT.
      4. Incubate in primary antibody cocktail (e.g., rat α-BrdU (bromodeoxyuridine), guinea pig α-DCX (doublecortin), goat α-GFP (green fluorescent protein)) diluted in TBSplus, O/N at 4°C. Rinse 3 times in TBS for 15 min each.
      5. Incubate in a cocktail of fluorochrome-conjugated secondary antibodies (e.g., Rhodamine Red α-rat, Alexa-647 α-guinea pig, Alexa-488 α-goat; all derived in donkey) diluted in TBSplus, 3 hr at RT or O/N at 4 °C. From now on protect sections from light. Rinse 3 times in TBS for 15 min each.
      6. Mount sections to slides in gelatin and air dry overnight (O/N). Coverslip with aqueous mounting medium.
   2. Sequential multiple immunofluorescence with primary antibodies from the same host species
      1. As steps 2.2.1.1 – 2.2.1.3.
      2. Incubate in first primary antibody (e.g., rabbit α-antigen A), O/N at 4 °C. Rinse 3 times in TBS and once in TBS-T for 10 min each.
      3. Incubate in first fluorochrome-conjugated secondary antibody (e.g., Rhodamine Red-conj. donkey α-rabbit), 3 hr RT. From now on, protect sections from light. Rinse 3 times in TBS and once in TBS-T for 10 min each.
      4. Incubate in 10% normal serum from the same host as the primary antibodies (e.g., rabbit serum) for 3 hr RT to saturate open paratopes on the first secondary antibody. Rinse 3 times in TBS and once in TBS-T for 10 min each.
      5. Incubate in TBSplus with 50 µg/ml unconjugated monovalent Fab fragments directed against the host of the primary antibodies (e.g., α-rabbit IgG (H+L)) to cover epitopes that could be recognized by the second secondary antibody, O/N at 4 °C.
      6. Rinse at least 3 times in TBS and once in TBS-T for 10 min each. While transferring, briefly swab the mesh inserts containing the sections on a paper towel to remove any Fab leavings.
      7. Incubate in second primary antibody (e.g., rabbit α-antigen B), O/N at 4 °C. Rinse 3 times in TBS and once in TBS-T for 10 min each.
      8. Incubate in second fluorochrome-conjugated secondary antibody (e.g., Alexa-488-conj. donkey α-rabbit), 3 hr RT. Rinse 3 times in TBS for 15 min each, mount and coverslip as above.
         NOTE: To label antigens with antibodies from different host species, add them to step 2.2.2.2 and the respective secondary antibodies to step 2.2.2.3.