Close
  Indian J Med Microbiol
 

Figure 1: Examples of difficulties visualizing three-dimension structures. (a) 60 μ m-thick 13.5d mouse embryo section was stained for Ki67 (green) and CD31 (red). The brain area was imaged using a confocal microscope with × 40 objective. XYZ section view shows that while penetration of anti-CD31 antibody is complete, anti-Ki67 antibody only detected cells in the very superficial layer of cells. The stack had to be imaged with laser compensation in order to view the signal in deeper layers. (b) Sagittal serial sections of an adult mouse brain stained with 4',6-diamidino-2-phenylindole only, were manually aligned using AutoAligner. The snapshot from the software displays one section in green and its adjacent section in magenta. The two sections were manually aligned along the cortical outline (white arrows). When this region is well-aligned, one sees that the cortex has expanded (yellow arrow). Olfactory bulb, cerebellum, and brain stem regions have rotated around relative to the cortex and cannot be aligned well (red arrows). (c) Snapshot from AutoAligner showing adjacent breast tumor xenograft sections stained for CD31 clearly see that these two adjacent sections cannot be aligned in any meaningful way, suggesting that there was more than a 5 μ m gap between the two adjacent sections. (d) XZ view showing the aligned sections of seminiferous tubules stained for DAZL (green) shows the inconsistency in the DAZL staining across the sections. (e) Whole-slide view of serial sections that were mounted and stained on one slide with the anti-DAZL antibody (green). While discovery XT automated stainer gives much more reproducible and reliable results, this particular slides exhibits uneven staining across the slide. (f) Zoomed view of E9.5 mouse embryo stained for nuclear pHH3. Snapshot from AutoAligner overlaying the pHH3 staining from two adjacent sections (green and magenta) shows the difficulty in aligning small signals such as nuclear staining. Scale bars: A =50 μ m, B = 2 mm, C = 200 μ m, D = 50 μ m, E = 5 mm, F = 100 μ m

Figure 1: Examples of difficulties visualizing three-dimension structures. (a) 60 μ m-thick 13.5d mouse embryo section was stained for Ki67 (green) and CD31 (red). The brain area was imaged using a confocal microscope with × 40 objective. XYZ section view shows that while penetration of anti-CD31 antibody is complete, anti-Ki67 antibody only detected cells in the very superficial layer of cells. The stack had to be imaged with laser compensation in order to view the signal in deeper layers. (b) Sagittal serial sections of an adult mouse brain stained with 4',6-diamidino-2-phenylindole only, were manually aligned using AutoAligner. The snapshot from the software displays one section in green and its adjacent section in magenta. The two sections were manually aligned along the cortical outline (white arrows). When this region is well-aligned, one sees that the cortex has expanded (yellow arrow). Olfactory bulb, cerebellum, and brain stem regions have rotated around relative to the cortex and cannot be aligned well (red arrows). (c) Snapshot from AutoAligner showing adjacent breast tumor xenograft sections stained for CD31 clearly see that these two adjacent sections cannot be aligned in any meaningful way, suggesting that there was more than a 5 μ m gap between the two adjacent sections. (d) XZ view showing the aligned sections of seminiferous tubules stained for DAZL (green) shows the inconsistency in the DAZL staining across the sections. (e) Whole-slide view of serial sections that were mounted and stained on one slide with the anti-DAZL antibody (green). While discovery XT automated stainer gives much more reproducible and reliable results, this particular slides exhibits uneven staining across the slide. (f) Zoomed view of E9.5 mouse embryo stained for nuclear pHH3. Snapshot from AutoAligner overlaying the pHH3 staining from two adjacent sections (green and magenta) shows the difficulty in aligning small signals such as nuclear staining. Scale bars: A =50 μ m, B = 2 mm, C = 200 μ m, D = 50 μ m, E = 5 mm, F = 100 μ m