An Immunofluorescence Staining Technique to Detect Adult Hippocampal Neurogenesis

Published: April 30, 2024

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…

開示

The authors have nothing to disclose.

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

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記事を引用
An Immunofluorescence Staining Technique to Detect Adult Hippocampal Neurogenesis. J. Vis. Exp. (Pending Publication), e22108, doi: (2024).

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