Immunohistochemistry and Multiple Labeling with Antibodies from the Same Host Species to Study Adult Hippocampal Neurogenesis

Summary

This video article illustrates a comprehensive protocol to detect and quantify all stages of adult hippocampal neurogenesis within the same tissue section. We elaborated a method to overcome the limitations of indirect multiple immunofluorescence that arise when suitable antibodies from different host species are unavailable.

Abstract

Adult neurogenesis is a highly regulated, multi-stage process in which new neurons are generated from an activated neural stem cell via increasingly committed intermediate progenitor subtypes. Each of these subtypes expresses a set of specific molecular markers that, together with specific morphological criteria, can be used for their identification. Typically, immunofluorescent techniques are applied involving subtype-specific antibodies in combination with exo- or endogenous proliferation markers. We herein describe immunolabeling methods for the detection and quantification of all stages of adult hippocampal neurogenesis. These comprise the application of thymidine analogs, transcardial perfusion, tissue processing, heat-induced epitope retrieval, ABC immunohistochemistry, multiple indirect immunofluorescence, confocal microscopy and cell quantification. Furthermore we present a sequential multiple immunofluorescence protocol which circumvents problems usually arising from the need of using primary antibodies raised in the same host species. It allows an accurate identification of all hippocampal progenitor subtypes together with a proliferation marker within a single section. These techniques are a powerful tool to study the regulation of different progenitor subtypes in parallel, their involvement in brain pathologies and their role in specific brain functions.

Introduction

Two brain regions constitutively generate new neurons throughout life, the subventricular zone of the lateral ventricles and the subgranular zone (SGZ) of the hippocampal dentate gyrus (DG). The newborn neurons derive from neural progenitor cells and go through different stages of morphological and physiological development before reaching maturity^1,2. From a slowly dividing radial glia-like stem cell (type 1) consecutive stages of transit amplifying intermediate progenitor cells arise. The more undifferentiated subtypes (type 2a and type 2b) have an irregular shape with short, tangential processes. They generate neuroblasts (type 3) that gradually exit the cell cycle to become immature neurons (with dendrites extended towards the molecular layer) and finally integrate into the hippocampal network as mature granule cells. Due to their particular physiological characteristics these cells provide the circuitry with enhanced plasticity³ suggesting a unique role in hippocampal function. Actually, studies of the last decade generated substantial evidence that adult neurogenesis contributes to spatial memory, pattern separation and emotional behavior^4,5.

Adult neurogenesis can be studied using different approaches. Thymidine analogs incorporate into DNA during the S-phase of the cell cycle and allow birth dating, quantification and fate analysis of newborn cells^6-8. Sequential application of different thymidine analogs (e.g., CldU, EdU or IdU) can be used to study cell turnover or cell populations born at different time points during the course of an experiment⁹. An alternative, endogenous marker for cell proliferation is Ki67. It is expressed in dividing cells during all phases of the cell cycle (G1, S, G2, M) except the resting phase (G0) and the beginning of G1 ^10,11. To analyze the phenotype of newborn cell populations in the adult dentate gyrus several stage-specific molecular markers can be used such as GFAP, nestin, DCX and NeuN^1,6. GFAP is a marker of mature astrocytes but is also expressed in radial glia-like cells in the adult forebrain. Nestin is an intermediate filament specific for radial glia-like cells and early intermediate progenitor cells. DCX is a microtubule-associated protein expressed in intermediate progenitors, neuroblasts and immature neurons. Based on the (co-) expression of these three markers and the morphological features of the labeled cells four distinct progenitor cell subtypes can be identified: type 1 (GFAP⁺, nestin⁺, DCX^-), type 2a (GFAP^-, nestin⁺, DCX^-), type 2b (GFAP^-, nestin⁺, DCX⁺) and type 3 (GFAP^-,nestin^-, DCX⁺)¹. Co-labeling of DCX together with NeuN, which is expressed in postmitotic neurons, allows the differentiation of immature (DCX⁺, NeuN⁺) and mature (DCX^-, NeuN⁺) granule neurons.

The above mentioned markers are frequently used for immunofluorescent co-labeling and subsequent confocal microscopy to analyze the number and identity of newborn cells. This typically requires antibodies from different host species to prevent undesired antibody cross-reactivity. However, the majority of primary antibodies suitable for neurogenesis research are raised either in rabbits or mice (e.g., mouse α-BrdU, mouse α-NeuN, rabbit α-Ki67, rabbit α-GFAP). This leads to serious limitations in the number and combination of antigens that could be evaluated in a single slice. This in turn not only increases the staining effort, as multiple stainings have to be performed, but might also compromise the reliability of results. Furthermore, some antigens are susceptible to formalin fixation-induced epitope masking (e.g., Ki67, nestin). We herein describe modifications from the classical single- and multiple immunolabeling protocols (e.g., epitope retrieval, multiple sequential immunostaining, use of nestin-GFP transgenic mice¹²) that overcome many of these issues. In particular, the sequential multiple immunofluorescence protocol allows staining against up to four different antigens even if part of the antibodies is derived from the same host. This enables the simultaneous detection of type 1, type 2a, type 2b and type 3 progenitor cells, as well as their proliferative activity within a single section.

Protocol

NOTE: All procedures involving living animals were carried out in accordance with the EC directive 86/609/EEC guidelines on the care and use of laboratory animals and approved by the local ethics committee (Thüringer Landesamt für Lebensmittelsicherheit und Verbraucherschutz).

1. Intraperitoneal Injection of Thymidine Analogs

1. Weigh animals the day before injection. Calculate the amount of thymidine analog required for all injections planned on the next day as well as individual weight-adjusted injection volumes of a 10 mg/ml stock solution.
2. Prepare 10 mg/ml thymidine stock solution. (CAUTION! Thymidine analogs are toxic. Follow the specific Material Safety Data Sheets (MSDS) provided by the suppliers, i.e., wear lab coat and gloves, use a chemical fume hood).
   1. Take thymidine analog from the freezer and bring it to RT, approx. 21 °C. Weigh 10 mg and add sterile saline (for BrdU and CldU) or 0.04 N NaOH (in sterile saline; for IdU), vortex. Place for at least 10 - 15 min in a 50 °C water bath and vortex every 2 - 3 min to dissolve the powder.
      NOTE: Use solutions for up to 24 hr when stored at RT and for several weeks at -20 °C. Protect solutions from light (cover with aluminum foil). Always check for precipitates and re-dissolve if necessary.
3. Restrain the mouse by the scruff and intraperitoneally inject an appropriate, weight-adjusted volume of stock solution (at RT) using a fine dosage syringe with a 30 G needle.
   NOTE: In case CldU and IdU have to be administered sequentially within the same animal, consider to inject equimolar concentrations (e.g., 42.5 mg/kg CldU and 57.5 mg/kg IdU, which correspond to 50 mg/kg BrdU). Therefore, adjust the injection volumes of the 10 mg/ml stock solutions accordingly.

2. 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 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.

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).

3. 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 ABC reaction 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 TBS and rinse thoroughly (once O/N at 4 °C, 5 times at RT for 10 min each) to completely remove antifreeze.
   2. Incubate for 30 min in 1.5% H₂O₂ in TBS-T to quench endogenous peroxidase activity. 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. Incubate sections for 30 min at 37 °C in 2 N HCl to denature DNA. 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 to block unspecific antibody binding sites, 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, 3 hr 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 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 lab coat and gloves, 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 peroxidase reaction. Incubate for 12 min. Rinse 3 times in TBS for 15 min each.
   11. Mount sections to slides in gelatin, air dry O/N. Coverslip with permanent mounting medium.
   12. Optional: Counterstain before placing the coverslip (see section 3.5).
       NOTE: If the signal-to-noise ratio is low because of high background, repeat H₂O₂ treatment after the incubation with AB reagent (step 3.1.8).
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 completely remove antifreeze.
      2. Optional: as steps 3.1.3 - 3.1.4.
      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, guinea pig α-DCX, goat α-GFP) diluted in TBSplus, O/N at 4°C. Rinse 3 times in TBS for 15 min each.
      5. Incubate in 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, air dry O/N. Coverslip with aqueous mounting medium.
   2. Sequential multiple immunofluorescence with primary antibodies from same host species
      1. As steps 3.2.1.1 - 3.2.1.3.
      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 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 3.2.2.2 and the respective secondary antibodies to step 3.2.2.3.
   3. One of the secondary antibodies from same species as one of the primary antibodies
      NOTE: This protocol is suitable for quadruple-staining against BrdU or Ki67 together with GFAP, nestin-GFP and DCX.
      1. Follow strictly protocol steps 3.2.1.1 - 3.2.1.5. Until this point use only donkey serum in TBSplus.
      2. Incubate in TBSplus containing 3% goat serum for 1 hr at RT. This covers open paratopes on the α-goat secondary antibody. Rinse 3 times in TBS and once in TBS-T for 10 min each.
      3. Incubate in AMCA-conj. goat α-rabbit diluted in TBSplus, 3 hr RT or O/N 4 °C. Rinse three times in TBS for 15 min each, mount and coverslip as above.
   4. CldU, IdU co-staining
      1. Rinse, denature and neutralize sections as described in section 3.2.1 (steps 3.2.1.1 - 3.2.1.2).
      2. Incubate with 20 µg/ml unconjugated Fab fragments α-mouse IgG (H+L) in TBSplus, 1 hr at RT. Rinse 4 times in TBS and once in TBS-T for 10 min each.
      3. Incubate in primary antibody cocktail containing rat α-BrdU (1:400; purified IgG2) and mouse α-BrdU (1:350) diluted in TBSplus, O/N at 4°C. Rinse 3 times in TBS and once in TBS-T for 10 min each.
      4. Incubate in secondary antibody cocktail containing biotinylated donkey α-rat (1:500) and FITC-conj. donkey α-mouse Fab fragments (1:100). Rinse 3 times in TBS and once in TBS-T for 10 min each.
      5. Incubate in Rhodamine Red-conj. Streptavidin, 2 hr at RT. Rinse 3 times in TBS for 15 min each, mount and coverslip as above.
   5. Amplification of fluorescence signal in nestin-GFP mice
      1. Add goat α-GFP to the primary antibody cocktail.
      2. Add a fluorochrome conjugated secondary antibody with spectral properties similar to GFP (such as Alexa 488 conj. donkey α-goat) to the secondary antibody cocktail.
3. Epitope retrieval
   1. Carry out epitope retrieval after rinsing out antifreeze. Preheat steamer with 6- or 12-well plates containing 0.1 M citrate buffer pH 6.0 to 95 - 99 °C (approximately 25 min).
   2. Transfer the sections into the hot citrate buffer and steam for 30 min (cover the plates with aluminum foil as plastic lids are not heat-resistant).
   3. Immediately place the plates in an ice bath to cool down. This helps to protect tissue morphology, which is of particular importance when working with brain sections of postnatal animals.
   4. Rinse 3 times in TBS for 10 min each to rinse out and neutralize the citrate and continue staining.
4. Mouse antibodies on mouse tissue
   1. Add 20 µg/ml monovalent Fab fragments α-mouse IgG (H+L; same host-species than secondary antibody) to the first blocking step (e.g., step 3.1.5 or 3.2.1.3).
   2. Rinse 4 times in TBS and once in TBS-T for 10 min each, and proceed with respective protocol.
5. Cresyl violet counterstaining
   1. Preheat cresyl violet solution to 60 °C (in glass jar). Incubate slides in the hot solution for 3 min.
   2. Rinse in aq. dest. and dehydrate 2 times for 1 min each in 70%, 96% and 100% isopropanol.
   3. Clear for 5 - 6 min in xylene or a xylene substitute and coverslip with a compatible permanent mounting medium.

4. Data Analysis

1. Count peroxidase stained newborn cells in every 6^th section along the entire rostrocaudal extent of the dentate gyrus. Use a light microscope at 400X magnification.
2. Multiply the resulting cell numbers with the intersection interval to obtain an estimate of total numbers of newborn cells.
3. Image the fluorescence labeled sections with a confocal laser microscope equipped with appropriate lasers and filter systems. Take image stacks at random positions along the entire extent of the dentate gyrus at 400X magnification and analyze at least 50 randomly selected cells of interest per hemisphere for co-labeling with other markers.
4. Multiply the resulting percentages of co-labeled cells (%/100) with the total numbers of newborn cells to calculate absolute numbers of specific newborn cell populations.

Results

We applied the methods described above to quantify and characterize newborn cells in the postnatal and adult hippocampus. Therefore, we used wildtype and neurogenesis-deficient cyclin D2 knock out (Ccnd2KO) mice housed under conditions known to affect the rate of neurogenesis (i.e., enriched environment, EE)^13,14. Immunohistochemical DAB staining against either Ki67, BrdU, CldU or IdU consistently revealed differences in newborn cell numbers between wildtype and Ccnd2KO mice (

Discussion

Quantification and identification of subpopulations of newborn cells is a central issue in adult neurogenesis research. Combining proliferation markers and antibodies against proteins expressed during specific stages of adult neurogenesis allows immunohistochemical detection of these subpopulations. Some of the antibodies or antibody combinations require specific staining conditions.

Labeling of dividing cells with synthetic thymidine analogs is still the gold standard for studying adult hippo...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Thymidine analog administration
5-Bromo-2′-deoxyuridine, BrdU	Sigma-Aldrich	B9285	toxic (mutagenic, teratogenic)
5-Chloro-2′-deoxyuridine, CldU	Sigma-Aldrich	C6891	toxic (mutagenic, teratogenic)
5-Chloro-2′-deoxyuridine, CldU	MP Biomedicals	2105478	toxic (mutagenic, teratogenic)
5-Iodo-2′-deoxyuridine, IdU	MP Biomedicals	2100357	toxic (mutagenic, teratogenic)
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	Novocastra/ Leica Biosystems	NCL-L-Ki67MM1	DAB 1:400/IF 1:100; requires epitope retrieval
Rabbit α-GFAP, AS-3-GF	Synaptic Systems	173 002	1:500
Goat IgG (H+L) α-GFP	Acris Antibodies	R1091P	1:300
Mouse IgG1 α-nestin	Abcam	ab6142	1:200; requires epitope retrieval
Guinea pig IgG (H+L) α-Doublecortin	Merck Millipore	AB2253	1:500
Rat IgG2a α-BrdU (ascites)	AbD Serotec/ Bio-Rad	OBT0030CX	for detection of BrdU; DAB 1:500/IF 1:400
Rat IgG2a α-BrdU (purified)	AbD Serotec/ Bio-Rad	OBT0030	for detection of CldU; DAB 1:500/IF 1:250-400
Mouse IgG1 α-NeuN	Merck Millipore	MAB377	1:500
mouse IgG1κ α-BrdU	BD Biosciences	347580	For detection of IdU; DAB 1:500/IF 1:350
Secondary antibodies
Donkey α-guinea pig IgG (H+L)-Biotin	Dianova	711-065-152	1:500
Donkey α-rat IgG (H+L)-Biotin	Dianova	712-065-150	1:500
Donkey α-mouse IgG (H+L)-Biotin	Dianova	715-065-151	1:500
Goat α-rat IgG (H+L)-Alexa Fluor 488	Molecular Probes	A11006	1:250
Donkey α-goat IgG (H+L)-Alexa Fluor 488	Molecular Probes	A11055	1:250
Donkey α-mouse IgG (H+L)-FITC, Fab-Fragment	Dianova	715-097-003	1:100
Donkey α-mouse IgG (H+L)-Alexa Fluor 647	Dianova	715-605-151	1:250
Donkey α-guinea pig IgG (H+L)-Alexa Fluor 647	Dianova	706-605-148	1:250
Donkey α-rat IgG (H+L)-Rhodamine Red-X	Dianova	712-295-150	1:250
Donkey α-rabbit IgG (H+L)-Rhodamine Red-X	Dianova	711-295-152	1:250
Donkey α-guinea pig IgG (H+L)-Rhodamine Red-X	Dianova	706-296-148	1:250
Streptavidin-rhodamine Red-X	Dianova	016-290-084	1:500
Goat α-rabbit IgG (H+L)-AMCA	Dianova	111-155-144	1:250, works only with rabbit α-GFAP
Hoechst 33342	Molecular Probes	H3570	1:1000
DAPI	Molecular Probes	D1306	1:1000
Blocking
Fab-fragment donkey α-mouse IgG (H+L)	Dianova	715-007-003	1:20
Fab-fragment donkey α-rabbit IgG (H+L)	Dianova	711-007-003	1:20
Normal donkey serum	Merck Millipore	S30
Normal rabbit serum	Dianova	011-000-010
Normal goat serum	Dianova	005-000-001
Bovine serum albumin	Sigma-Aldrich	A7906
Histology
Cresyl violet	Sigma-Aldrich	C5042
Neo-Clear	Merck Millipore	109843	non-toxic xylene substitute
Neo-Mount	Merck Millipore	109016	permanent mounting medium
Microscopy
Axioskop 2	Carl Zeiss Microscopy
LSM 710	Carl Zeiss Microscopy