C57BL/6 mice were given three intraperitoneal (IP) injections of BrdU (60 mg/kg body weight, 15 mg/ml BrdU solution pH 7.2) at two hour intervals and then were then sacrificed at various times after the final BrdU injection (12 hours, 7 days, 14 days, 20 days, 40 days and 75 days). Mice were anesthetized by IP injection of 0.1%/0.1% ketamine/xylazine dissolved in water. Once the animals were non-responsive, they were perfused transcardially with 1% NaCl in 0.1 M phosphate buffer (PB) followed by 4% paraformaldehyde (Sigma) in PB. The brains were extracted and post fixed in 4% paraformaldehyde in PB overnight and then sunk in 30% sucrose in PB. Each hemisphere of the brains was cryogenically sliced into 25 micron sagittal sections using a sliding microtome (Leica SM2000R).
Double labeling immunofluorescence
Sections from the central 60% of the olfactory bulb (the part containing accessory olfactory bulb) were used for double immunofluorescence labeling. Separate analysis of the medial and lateral most sections revealed no significant differences in the fractions of cells double labeled and so data from all sections were combined for subsequent analysis (fractions of double positive cells in lateral-most, medial-most sections and overall: CR 9 ± 1%, 10 ± 1%, 9 ± 1%; NC 22 ± 5%, 22 ± 3% and 22 ± 2%). Sections from each animal were used for each combination of antibodies, which consisted of anti-BrdU and one of the following: anti-calbindin (CB), anti-calretinin (CR), anti-GABA, anti-N-copine (NC), anti-tyrosine hydroxylase (TH), or anti-NeuN (NeuN). In addition, NeuN was used in combination with anti-S100β (S100B). Sources and dilutions of all antibodies used can be found in Table 1. All incubations were done at room temperature with oscillation. Sections to be labeled with BrdU were first incubated with 2 M HCl for 1 hour to denature the DNA, followed by 2 brief rinses with PB. To permeabilize and block the tissue, the sections were incubated in 0.1% Triton X-100 (Sigma) and 2% normal donkey serum (NDS) (Jackson Immuno Research Laboratories) in PB for 1 hour. The sections were washed 3 times with PB for 10 minutes each wash with oscillation. This wash procedure was also performed following each of the antibody incubations. The antibodies were added in concentrations according to Table 1 in 2% NDS in PB and incubated for 1 hour. The BrdU primary antibody was added followed by the fluorescent BrdU secondary antibody emitting at a red wavelength. Then one of the other primary antibodies was added followed by the corresponding fluorescent secondary antibody emitting at a green wavelength (Table 1). In the case of the combination of S100B and NeuN, the S100B primary antibody was the first in the labeling sequence, and was followed by the fluorescent S100B secondary antibody emitting at a green wavelength, followed by NeuN and its corresponding red secondary antibody. At the addition of the BrdU secondary antibody, all subsequent incubations were performed in the dark. The sections were mounted on collagen coated slides using gelvatol as the mounting medium.
Neuro Trace labeling
Sections from the central half of the olfactory bulb were co-labeled with the fluorescent Nissl stain NeuroTrace 530/615 red (NTr) or NeuroTrace 500/525 (NTg). (Molecular Probes) and one of BrdU, CB, CR, GABA, NC, or TH. As the only difference between NTr and NTg is the fluorescent color, both stains will be referred to as NT In addition, NT was used to compare NeuroTrace labeling to that of NeuN. or anti-S100B. Four sections from each animal were used. All incubations were done at room temperature with oscillation, and all wash steps consisted of three 10 minute incubations in PB, except where noted. The sections were permeabilized and blocked by incubation in 0.1% Triton X-100 and NDS, and then washed. Tissue to be stained with BrdU was then treated with 25 units/mL of DNAse I (Sigma) in 5 mM MgCl2 in PB for 1 hour at 37°C with no oscillation, and then rinsed briefly twice with PB. DNAse was used instead of HCl to denature the DNA because NT staining was not seen in tissue subjected to the HCl wash. In control experiments we observed that while DNAse resulted in less intense staining of BrdU positive cells, similar numbers of cells were BrdU positive in DNAse and HCl treated tissue and the fraction of cells colabeling with secondary markers was similar in the two conditions. All primary and secondary antibody labeling (CB, CR, GABA, NC, TH, NeuN, and S100B) was done as described in the Double Labeling Immunofluorescence section. CB, CR, GABA, N-C, TH, and S100B were labeled with a fluorescent secondary emitting at a green wavelength whereas BrdU and NeuN were labeled with a fluorescent secondary emitting at a red wavelength, (Table 1). Sections were then incubated with 1:100 NTg or NTr, as appropriate, in PB for 20 min, and then washed once with 0.1% TX-100 in PB for 10 min, twice more with PB only for 10 min, followed by a final wash step of PB for two hours. Sections were then mounted on collagen coated slides using gelvatol as the mounting medium.
Microscopy and image processing
For all sections, 12 bit grayscale images were acquired using an Olympus BX51 microscope fitted with an Optronics Microfire camera and a Ludl x-y-z motorized stage, and mounted on a tabletop vibration isolation table (TMC).
For imaging granule cells stained with CB and CR, two series of images consisting of the entire olfactory bulb were taken with a 10× objective (NA = 0.4). One series visualized the BrdU labeling, and one visualized the CB or CR labeling. These image series were acquired automatically and automatic digital montages were made using the virtual slice module of Neurolucida (Microbrightfield). Exposure times were selected to avoid image saturation. The glomerular region in all virtual slice images was manually masked using Adobe Photoshop, and then the double labeled cells in each image pair were counted using custom software written in Igor Pro (Wavemetrics). Before counting, images underwent background subtraction using a median filtered image, followed by image thresholding to produce a binary image. The threshold value was 5 standard deviations above the mean pixel value of the image. After thresholding, the image underwent contraction and dilation operations to eliminate isolated "noise" pixels. Areas of multiple above-threshold pixels were then identified and their area determined. Groups of connected pixels greater than 9.12 microns (12 pixels) in area were counted as "cells" in single images. This is the approximate area of granule cell nuclei. For counting double positive cells, we combined the binary images taken in the two color channels and created images in which only pixels that were above threshold in both of the single images were above threshold. These images were then counted as above to determine the number of double positive cells. The validity of this process was checked in several control experiments in which the same primary antibody was labeled with two different color secondary antibodies and in other experiments in which two different primary antibodies, directed against the same antigens but made in different species, were used to stain the same tissue. In these experiments our automated double labeling technique was able to identify ~95% of the cells as double positive with a false-positive rate estimated to be less than 2%.
For other antibodies that had higher background staining, and for all glomerular layer imaging, image pairs were acquired using a 40× oil immersion objective (NA = 1.0). For each section, four sets of 40× images of the GCL were acquired, two from the deep GCL and two from the superficial GCL. Four sets of 40× images of the GLL were obtained at even intervals over the GLL. To avoid bias in the selection of regions, we identified cells in the BrdU image, optimized the focus, and then the subsequent CB, CR, GABA, NC, or TH image was obtained without changing the focal plane. Images were combined such that the BrdU images were in the red channel and the appropriate second label (CB, CR, GABA, N-C, or TH) images were in the green channel. These combined images were manually counted by identifying BrdU positive cells, and then overlaying the green channel to determine colocalization of the second label. Cells were identified as double positive only if both the red and green elements were in focus and were much brighter than the background.
For NeuroTrace or NeuN labeled sections, 40× images of the GLL and GCL were acquired as described above. Manual counting of the images was done by visualizing the NT or NeuN channel only; 10 NT or NeuN positive cells were picked at random, and then the co-label channel was overlaid onto the NT or NeuN channel. Cells were identified as double positive as described above.
For all double labeling experiments we took care to avoid counting as double labeled bits of autofluorescent material that appeared frequently but randomly in the glomerular layer. This material, which was also seen in unlabelled tissue, in some cases accounted for a large fraction of the fluorescent objects seen in the glomerular layer, and was very broadly fluorescent, producing bright signals across all filter sets. (See supplemental figure 1). Thus, such spots with strong fluorescence across DAPI, FITC, and Rhodamine filter sets were not counted as being double positive.
In a subset of sections from several animals we compared double counts from epifluorescence images with counts of double labeled cells from confocal images. Immunolabeled sections stained (as above) for CR, N-C and GABA were analyzed using a laser-scanning confocal microscope (LSM 510, Zeiss) and 25× oil objective (Plan-NEOFLAUR, Zeiss). All BrdU labeled cells in a given volume of the granule cell layer were counted by visual inspection with care taken to identify multiple labeled cells in the Z-axis. Cells co-labeled for BrdU and Double labeled cells were subsequently visually identified in the population of BrdU labeled cells. Tissue was analyzed from 4 separate animals for each cellular marker. No significant differences were observed in the fraction of double labeled cells measured by these two different approaches (see Figure 4 and additional file 2).
For all statistical comparisons, we calculated the fraction of double labeled cells across all sections taken from a single animal and we then computed the mean and standard error of these fractions across animals. In all cases significance was computed across groups of animals subject to the same condition.