The Journal of Neuroscience, May 30, 2007, ():
Antigen-Retrieval Procedure for Bromodeoxyuridine Immunolabeling with Concurrent Labeling of Nuclear DNA and Antigens Damaged by HCl Pretreatment
J. Neurosci. Tang et al.
27: 5837
Supplemental Data
Files in this Data Supplement:
- supplemental material
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Supplemental Figure 1. The morphology of tissue pretreated by the steamer/citrate method is equal or superior to the morphology of tissue pretreated with HCl.
Parasagittal sections of P1 rat brain were pretreated using the standard steamer/citrate (A, C) or HCl protocols (B,D); developed with anti-BrdU, anti-Type III ?-tubulin, and Hoechst (see Materials and Methods for details); and examined at low power (Zeiss Axioscope; 10X lens; A,B) and high power (Zeiss Axioplan equipped with the Zeiss LSM 510 confocal system; 63X lens; C,D). For this Figure we did not use identical imaging parameters and processing for the steamer/citrate and HCl pretreated tissues but rather optimized the processing for each pretreatment. This required using longer exposure times (B) and higher laser intensities (D) for HCl pretreated tissue as compared to steamer/citrate pretreated tissue (A,C). Morphological details, such as fine processes, are well preserved. The box in A4 outlines the field shown in C; the box in B4 outlines the field shown in D. This boxed area includes a portion of the RMS-vl and, along the left side of the box, the extension of the lateral ventricle toward the olfactory bulb. Neural cell processes (arrows in C2 and D2) are as well, or better defined, in the steamer/citrate pretreated tissue compared to the HCl pretreated tissue. Scale bars: B4, 200 ?m (applies to A – B); D3, 20 ?m (applies to C – D).
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Supplemental Figure 2. The specificity of BrdU antibody labeling is preserved in steamer/citrate pretreated tissue.
As noted in Materials and Methods, when tissue from an animal not injected with BrdU was labeled using the BrdU antibody following either steamer/citrate or HCl pretreatment, no nuclear BrdU signal was observed (not shown). As a further test of the specificity of BrdU antibody labeling of steamer/citrate processed tissue, parasagittal sections of P1 rat brain were pretreated using the standard steamer/citrate protocol; developed with anti-BrdU, anti-NeuN (Chemicon MAb377), and Hoechst; and examined at low (10X lens; A,B) and high power (60X lens; C). The box in B4 outlines the field shown in C. NeuN is a protein expressed by postmitotic neurons. Due to the short survival time following BrdU injection (3 h), it is expected that no NeuN(+) neurons will also be BrdU(+). As predicted, no double-labeled cells were observed in the SVZa region (A) or the olfactory bulb (B, C). Scale bars: B4, 200 ?m (applies to A – B); C4, 40 ?m (applies to C).
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Supplemental Figure 3. The steamer/citrate method can be used for BrdU immunolabeling free-floating tissue.
For these experiments P1 rats were perfused, and the brains postfixed as described in Materials and Methods. Parasagittal sections (20 um) were prepared on a freezing, sliding microtome then stored in 0.1M PBS at 4°C until their use. 350 to 400 ?l of the 10 ?M citrate buffer (pH 6.0) was applied to a standard microscope slide (not SuperFrost). Following rinsing in PBS, the sections were transferred with a brush to the pool of buffer on the slide, and carefully unfolded using the brush. The slide was then heat-treated in the steamer as described in Materials and Methods for the cryostat sections. Subsequent steps were performed with the sections on the slide. Throughout all steps, including the steamer treatment, the section floated in the incubation solution. This allowed solution access to both sides of the section but at the same time conserved reagents compared to returning the section to a dish. With the exception that the sections were floating, the immunostaining protocol used was identical to that used for cryosections. Following the last wash, excess PBS was carefully removed with a pipettor, the section was allowed to dry onto the slide, and the slides were coverslipped with VectaShield. Solid lines demarcate the border of the lateral ventricle. Steamer/citrate and HCl pretreated sections were immunolabeling and imaged in parallel using identical parameters. Scale bars: B4, 200 ?m (applies to A – B); D3, 20 ?m (applies to C – D).
