Serial histology and blockface images, mouse heart, 300 sections
<p>Mouse hearts were excised after Schedule 1 culling according to the UK Home Office guidance on the Operation of Animals (Scientific Procedures) Act of 1986, washed swiftly in 37°C normal Tyrode solution (NaCl 140 mM; KCl 5.4 mM; MgCl2 1 mM; HEPES 5 mM; Glucose 10 mM; CaCl2 1.8 mM; pH 7.4, 3...
| 總結: | <p>Mouse hearts were excised after Schedule 1 culling according to the UK Home Office guidance on the Operation of Animals (Scientific Procedures) Act of 1986, washed swiftly in 37°C normal Tyrode solution (NaCl 140 mM; KCl 5.4 mM; MgCl2 1 mM; HEPES 5 mM; Glucose 10 mM; CaCl2 1.8 mM; pH 7.4, 30010 mOsm) containing heparin (10u/ml) and then cannulated in cold Tyrode solution with heparin in order to perfuse the coronary vasculature from the aorta. Afterwards, hearts were arrested using modified Tyrode containing elevated potassium (20 mM), fixed by coronary perfusion with 10 mL of the fast-acting Karnovsky's fixative (0.75% formaldehyde, 0.75% glutaraldehyde mix) and stored overnight at 4°C. Hearts then were rinsed in cacodylate buffer (3×), dehydrated by exposure to rising alcohol concentrations (8 h in 25%, followed by 1.5 h in each of 50/70/80/90% alcohol, and 3 × 1.5 h in 100% alcohol), treated with xylene (3 x 1.5 h), infiltrated with wax (24 h in 50% and 48 h in 100%) and embedded in form of wax blocks. Wax blocks were mounted on Leica SM2400 heavy-duty sledge-type microtome and whole hearts were serially sectioned at 10 µm thickness. </p>
<p>Using remote-controlled Canon EOS 450D cameras mounted above the sledge, two photographs of the wax block surface were taken prior to each section (Figure 1), an approach developed by our group in the last years (Casero et al., 2016; Gruscheski et al., 2015; Siedlecka et al., 2013a, 2013b). The first photograph was taken at the Brewster angle for air/paraffin (55° to the surface normal). At this angle, the surface-vertical component of unpolarised incident light is totally transmitted, while most of the surface-horizontal component is reflected. A collection filter, aligned with the horizontal component filters out residual light refracted from lower tissue layers. Thus, the 55° image treats wax as a mirror and displays a negative of the topmost tissue layer, showing good delineation of tissue-wax boundaries. The second photograph was taken top-down (0° to the surface normal) as a reference to correct the perspective distortion in the 55° image.</p>
<p>After cutting, tissue sections were allowed to relax in a water bath (Leica Microsystems, HI 1210) at 39°C for several minutes, and then carefully mounted on positively charged slides (SuperFrost, VWR), aiming for minimal distortion and avoidance of tissue folds. Slides were air-dried overnight, followed by de-waxing and Picro Sirius red-staining, using a Leica AutoStainer XL, ST5010. The stained and coverslipped sections were imaged at high resolution (0.46 µm x 0.46 µm) with Hamamatsu Nanozoomer HT 2.0 and/or Zeiss Axio Scan.Z1 scanners, producing images between 8,862 x 6,643 (86M) and 28,672 x 23,296 pixels (1.2G) depending on tissue sample size.</p>
<p>Casero, R., Siedlecka, U., Jones, E., Gruscheski, L., Johnston, C., Grau, V., Kohl, P., 2016. Block-face imaging of wax-embedded tissue as a necessary step in the three-dimensional reconstruction of the heart structure, in: 82th Annual Meeting of the German Cardiac Society – Cardiac and Circulation Research, March 30. - April 2. - 2016, Mannheim. Springer Berlin Heidelberg, Mannheim, Germany, p. P1685. doi:10.1007/s00392-016-0967-z</p>
<p>Gruscheski, L., Jones, E., Johnston, C.M., Kohl, P., Siedlecka, U., 2015. Dual block-face imaging of wax-embedded whole heart on a motorized rotary microtome for improved serial two-dimensional histology stack acquisition. Proc. Physiol. Soc. Proc Physiol Soc 34.</p>
<p>Siedlecka, U., Casero, R., Burton, R.A., Afonso, C., Bollensdorff, C., Grau, V., Kohl, P., 2013a. Three-dimensional histological reconstruction of the heart guided by block-face imaging of wax-embedded tissue, in: 6th International Workshop on Cardiac Mechano-Electric Coupling and Arrhythmias, 12 Sep 2013 – 15 Sep 2013. MEC2013. p. #19.</p>
<p>Siedlecka, U., Casero Canas, R., Burton, R., Afonso, C., Bollensdorff, C., Grau, V., Kohl, P., 2013b. Block-face imaging of wax-embedded tissue as a modality to aid three-dimensional histological reconstruction of the heart, in: 37th Congress of IUPS (Birmingham, UK) (2013) Proc 37th IUPS. p. PCD033.</p> |
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