An Immunofluorescence Staining Technique to Detect Adult Hippocampal Neurogenesis

Abstract

Source: Ansorg, A., et al. Immunohistochemistry and Multiple Labeling with Antibodies from the Same Host Species to Study Adult Hippocampal Neurogenesis. J. Vis. Exp. (2015)

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 Prepare 4% formaldehyde in 0.1 M phosphate buffer pH 7.4 on the day before perfusion. Store at 4 °C. 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 (f…

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 nth section of an entire brain. For quantification of newborn cells, take every 6th…

Materials

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