An Immunofluorescence Staining Technique to Detect Adult Hippocampal Neurogenesis

Overview

The video illustrates an immunofluorescence staining method used to examine neurogenesis in the adult hippocampus. Initially, a mouse brain section undergoes treatment with primary antibodies directed at marker proteins associated with neurogenesis in neuronal progenitor cells. Next, it is labeled with secondary antibodies tagged with fluorophores. The brain section is then observed under a confocal microscope to distinguish various subtypes of progenitor cells, based on the expression patterns of distinct combinations of marker proteins.

Protocol

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.

Representative Results

Figure 1. Schematic illustration of transferring microtome slices into a 24-well plate. Start at A1 and place subsequent slices into row A; after A6, go to the next row B, and so forth. When reaching D6, go back to A1 and continue. This arrangement of slices allows for quantification of every n^th section of an entire brain. For quantification of newborn cells, take every 6^th brain section (equivalent to the content of one column); for immunofluorescence phenotyping, take every 12^th section (equivalent to the content of 2 alternating rows of one column)

Materials

Name	Company	Catalog Number	Comments
Tissue preparation
Isoflurane-Actavis	Piramal Healthcare	700211
Paraformaldehyde powder (PFA)	Riedel-De Häen	16005	toxic, flammable
Perfusion pump PD5206	Heidolph Instruments	523-52060-00
Masterflex Tygon lab tubing, Ø 0.8 mm	Thermo Fischer Scientific	06409-13
Feeding needle, straight, 21 G, 1.75 mm olive tip, 40 mm	Agnthos	1036
Freezing microtome Microm HM 400	Thermo Fischer Scientific
24-well Cell culture multiwell plates	Greiner Bio-One	662160
Immunohistochemistry
Tefal vitacuisine steamer	Tefal	VS 4001
Netwell 24 mm polyester mesh membrane inserts	Corning	3479	pre-loaded in 6-well culture plates
Netwell 15 mm polyester mesh membrane inserts	Corning	3477	pre-loaded in 12-well culture plates
Netwell plastic 6-well carrier kit	Corning	3521	for 24 mm polyester mesh membrane inserts
Netwell plastic 12-well carrier kit	Corning	3520	for 15 mm polyester mesh membrane inserts
Vectastain Elite ABC kit	Vector Laboratories	PK-6100
DAB (3,3′-Diaminobenzidine tetrahydrochloride hydrate)	Sigma-Aldrich	D-5637	carcinogenic, light sensitive
Fluoromount-G	SouthernBiotech	0100-01
Primary antibodies
rabbit IgG1 α-Ki67
rabbit α-GFAP, AS-3-GF	Novocastra/ Leica Biosystems	NCL-L-Ki67MM1	DAB 1:400/IF 1:100; requires epitope retrieval
goat IgG (H+L) α-GFP	Synaptic Systems	173 002	09:20
mouse IgG1 α-nestin	Acris Antibodies	R1091P	06:00
guinea pig IgG (H+L) α-Doublecortin	Abcam	ab6142	1:200; requires epitope retrieval
rat IgG2a α-BrdU (ascites)	Merck Millipore	AB2253	09:20
rat IgG2a α-BrdU (purified)	AbD Serotec/ Bio-Rad	OBT0030CX	for detection of BrdU; DAB 1:500/IF 1:400
mouse IgG1κ α-BrdU	AbD Serotec/ Bio-Rad	OBT0030	for detection of CldU; DAB 1:500/IF 1:250-400
mouse IgG1 α-NeuN	BD Biosciences	347580	for detection of IdU; DAB 1:500/IF 1:350
Secondary antibodies	Merck Millipore	MAB377	09:20
donkey α-guinea pig IgG (H+L)-Biotin
donkey α-rat IgG (H+L)-Biotin	Dianova	711-065-152	09:20
donkey α-mouse IgG (H+L)-Biotin	Dianova	712-065-150	09:20
goat α-rat IgG (H+L)-Alexa Fluor 488	Dianova	715-065-151	09:20
donkey α-goat IgG (H+L)-Alexa Fluor 488	Molecular Probes	A11006	05:10
donkey α-mouse IgG (H+L)-FITC, Fab-Fragment	Molecular Probes	A11055	05:10
donkey α-mouse IgG (H+L)-Alexa Fluor 647	Dianova	715-097-003	02:40
donkey α-guinea pig IgG (H+L)-Alexa Fluor 647	Dianova	715-605-151	05:10
donkey α-rat IgG (H+L)-Rhodamine Red-X	Dianova	706-605-148	05:10
donkey α-rabbit IgG (H+L)-Rhodamine Red-X	Dianova	712-295-150	05:10
donkey α-guinea pig IgG (H+L)-Rhodamine Red-X	Dianova	711-295-152	05:10
Streptavidin-Rhodamine Red-X	Dianova	706-296-148	05:10
goat α-rabbit IgG (H+L)-AMCA	Dianova	016-290-084	09:20
Hoechst 33342	Dianova	111-155-144	1:250, works only with rabbit α-GFAP
DAPI	Molecular Probes	H3570	17:40
Blocking	Molecular Probes	D1306	17:40
Fab-fragment donkey α-mouse IgG (H+L)
Fab-fragment donkey α-rabbit IgG (H+L)	Dianova	715-007-003	01:20
Normal donkey serum	Dianova	711-007-003	01:20
Normal rabbit serum	Merck Millipore	S30
Normal goat serum	Dianova	011-000-010
Bovine Serum Albumine	Dianova	005-000-001
Histology	Sigma-Aldrich	A7906
Cresyl violet
Neo-Clear	Sigma-Aldrich	C5042
Neo-Mount	Merck Millipore	109843	non-toxic xylene substitute
Microscopy	Merck Millipore	109016	permanent mounting medium
Axioskop 2
LSM 710	Carl Zeiss Microscopy
	Carl Zeiss Microscopy