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 |
