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11 protocols using rm2125 rotary microtome

1

Histological Analysis of Liver Tissues

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Liver tissue samples were withdrawn for further histological analysis. For the histopathologic investigation of the liver, the harvested samples were fixed in neutral 10% buffered formalin and were subsequently embedded in paraffin, sectioned at 4 μm thicknesses (Leica rotary microtome, RM2125, Nussloch, Germany) and stained with hematoxylin and eosin (H&E) and PAS methods (Periodic acid–Schiff staining). Histologic examination was performed with the aid of an Olympus BX51 microscope connected to a digital camera (Olympus DP-25, Tokyo, Japan). The microphotographs were acquired using an Olympus system for image acquisition and analysis (Olympus Cell B software, Tokyo, Japan).
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2

Histological Evaluation of Kidney and Liver Tissues

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Fragments of kidney and liver tissue were sectioned into 4 mm thick slices and preserved in 10% formalin for five days. They were subsequently treated with three 1-butanol baths followed by progressive dehydration with 70%, 96%, and 100% ethyl alcohol for one hour each. Following fixation, samples were prepared for histology by being embedded in paraffin, cut into 5 µm sections using a Leica rotary microtome (RM2125, Wetzlar, Germany), and stained with Goldner’s trichrome. An Olympus BX-41 microscope coupled with an Olympus E330 camera (Olympus, Tokyo, Japan) was used to evaluate the slides. The observed modifications were scored as follows:

Renal tissue:

0 = unmodified histological aspect
1 = zonal septal congestion with isolated vacuolar degenerative lesions
2 = zonal septal congestion, vacuolar degeneration, and isolated apoptosis of the tubular epithelium
3 = diffuse septal congestion, glomerular congestion, multifocal vacuolation, and apoptosis of the tubular epithelium
4 = coagulative necrosis of the tubular epithelium along with the presence of proteinaceous material in the lumen of the renal tubules
0 = unaltered liver parenchyma
1 = localized vacuolar degenerative lesions
2 = multifocal vacuolar degeneration with isolated hepatocyte apoptosis
3 = diffuse hepatocyte vacuolation throughout the liver lobule and isolated hepatocyte apoptosis
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3

Constructing Tissue Microarrays for Breast Cancer

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We made tissue microarrays (TMAs) of the breast tissue samples [12 (link)]. As described, the tissue cores were punched from donor blocks with a 1-mm-diameter stylus and loaded to recipient paraffin blocks. The design of the TMAs made it possible to evaluate the frequency of markers across tumor grades and molecular subtypes from patient tumors. The TMA layout on the recipient TMA blocks was designed in advance to include duplicates of each tissue sample and to organize the tissue samples randomly across the TMAs. Tissue regions were selected and included in the TMA based on the pathology of the tissue determined from the H&E-stained slides of each tissue block. The TMA blocks were constructed with a Veridiam Advanced Tissue Arrayer VTA-110CC. Blocks were sectioned at 3 μm thickness with Leica rotary microtome RM2125. Each of the two TMAs used for the staining had ~100 tissues per block with duplicates of each tissue sample included across the two TMAs.
For breast cancer tissue TMAs, including two or more tissue punches of 1-mm core diameter is preferred over a single punch or larger punch are more consistent and representative of the entire tumor [17 , 18 (link)].
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4

Histopathological Analysis of Tumor Samples

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The tumor samples collected were fixed in 10% buffered formalin for three days before going through a stepwise dehydration method (ethylic alcohol at 70%, 96%, and 100%) for one hour at a time. The clarification was performed in three 1-h-long 1-butanol baths before being embedded in paraffin. For histological analysis, 5 µm tissue sections were cut using a Leica rotary microtome (RM2125, Wetzlar, Germany) and stained with Goldner’s trichrome [32 ]. The slide’s evaluation was accomplished using an Olympus BX-41 microscope (Olympus, Tokyo, Japan) attached to the Olympus E330 camera (Olympus, Japan). Tumor samples were assessed for size, symmetry, delimitations, development, necrosis/ulceration, inflammatory infiltration, regression, cellular atypia, mitoses, melanization, and isolated cell proliferation.
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5

Quantifying Kidney Injury Histology

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Kidney samples were fixed in 4% neutral buffered formalin. Tissue slices were embedded in paraffin using a Thermo Shandon PathCentre tissue processor. 3-μm-thick sections were cut with a Leica RM2125 rotary microtome. After deparaffinization and re-hydration, the sections were routinely stained for haematoxylin-eosin (HE) and periodic acid Schiff (PAS). The extent of tubular cell edema, apical cytoplasm vacuolization, tubular cell vacuolization, tubular lumen irregularity, loss of brush border, sloughing of tubular cells, tubular dilation, tubular cell necrosis, flattened and simplified tubular epithelium, and denudement of tubular basal membrane was assessed by a renal pathologist in a blinded way. To quantify these changes, we set up a kidney injury score, based on the extent of the histological changes noted above (0 points = 0%; 1 point = 1–20%; 2 points = 21–40%; 3 points = 41–60%; 4 points = 61–80%; and 5 points = 81–100%). The values of the tubular cell necrosis and denuded basement membranes were weighted by a factor of two.
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6

Visualizing Arabidopsis Leaf Immunity

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Leaves of A. thaliana wild-type (Col-0) or the shrk1 x shrk2 double mutant were harvested at 5 dpi, cleared in 10% KOH for 5 min, stained with 0.05% aniline blue in 67 mM K2HPO4 for 20 min and observed with a Leica SP5 confocal light scanning microscope. Images were edited using ImageJ with the “volume viewer” plugin [77 (link)].
For Technovit sections, A. thaliana wild-type (Col-0) or mutant leaves were infected with Hpa as described above and harvested at 7 dpi. Leaves were fixed with 3.7% formaldehyde and dehydrated by incubating samples in 30%, 50%, 70% and 100% ethanol. Samples were embedded in Technovit 7100 according to the manufacturer’s instruction. A Leica RM2125 rotary microtome was used to cut 7 μm sections. Sections were stained in 0.01% trypan-blue-lactophenol for 3 h at 37°C, followed by clearing in chloral hydrate (2.5g/ml) and subsequent differential interference contrast microscopy with a Leica DMI6000B.
A. thaliana wild-type (Col-0) plants expressing RPW8.2-YFP [38 (link)] were infected with Hpa as described above, harvested at 10 dpi and observed with a Leica TCS SP 5 confocal laser scanning microscope equipped with a 63x NA 1.2 water-immersion objective (excitation with an Argon laser 514 nm, detection at 520–560 nm).
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7

Paraffin-Embedded Gynoecia Sectioning and Staining

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Tissues were fixed for 16 h at 25°C in 3.7% formaldehyde, 5% acetic acid, and 50% ethanol and subsequently dehydrated through an ethanol series until 70%. The tissues were embedded in paraffin. An RM 2125 rotary microtome (Leica) was used to make 10 µm transverse sections of Col, spt-12 and hat3 athb4 gynoecia at stage 12. Paraffin was removed from the sections by two rounds of incubation in 100% Histoclear (National Diagnostics) for 10 min at room temperature, followed by two washes in 100% ethanol for 2 min at room temperature, air-dried for 30 min and stained for 10 min by an aqueous solution containing 0.005% Toluidine blue O (Acros Organics). Slides were washed for 1 min in water; sections were mounted in a histological mounting medium Histomount (National Diagnostic) and examined under Leica DM600 light microscopy. Images were taken using the Leica LAS AF7000 software.
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8

Histological Analysis of Nile Tilapia Tissues

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The gills, liver, brain and muscles of Nile tilapia were fixed in 0.1 M phosphate‐buffered formaldehyde solution (pH 7.4). After trimming, the tissues were washed under slow running water for 24 h. Subsequently, they were dehydrated in a series of alcohols (70%, 80%, 90%, 96% and 100%) for varying durations, followed by immersion in xylene and xylene‐paraffin for 30 min each. The tissues were then incubated in soft paraffin (46–48°C) for 15 min and in hard paraffin (56–58°C) for 30 min before embedding in paraffin blocks using Leica EG 1150 H device. Sections of 4 µm thickness were obtained using a Leica RM2125 Rotary Microtome. The sections were stained using the Haematoxylin‐Eosin method and subsequently immersed in alcohol and xylene. The prepared sections were mounted on glass slides using Entellan Merck. The slides were examined under a light microscope (Leica DM‐750), and digital photographs were taken of the areas where lesions were observed (Culling et al., 2014 ).
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9

Tamoxifen-induced Lineage Tracing in Mist1-expressing Cells

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Mist1CreERT2;R26tdTomato mice were administered tamoxifen (0.25 mg/g body weight) by gavage daily for 4 days (Aure, et al., 2015b (link)). Mice (n = 3) were sacrificed for gland removal 3 days after the last tamoxifen gavage. Mist1CreERT2;R26tdTomato mice not treated with tamoxifen served as controls to detect non-specific recombination. SMG were fixed overnight in 4% PFA. For frozen sections, whole glands were transferred through a sucrose gradient (5%, 10%, 15% sucrose for 30 minutes each) and incubated in 30% sucrose : OCT (50:50) solution overnight. Samples were embedded in OCT and sectioned at 10 μm using a cryostat. For paraffin sections, SMG was processed in a Tissue-Tek VIPTM processing machine (Sakura Finetek USA, Inc.) and embedded in paraffin. Sections (5 μm) were cut using a Leica RM2125 rotary microtome.
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10

Automated Tissue Processing and Staining

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An automated tissue processer (Tissue-Tek® VIP™ Vacuum In ltration Processor) was used for tissue processing, which runs for a total time of ± 12 hours according to the protocol in Supplementary Table S1. Following sectioning of blocks (using the Leica RM 2125 rotary microtome (SMM instruments, Germany)), tissue sections were transferred to a water bath and placed onto standard microscope slides for haematoxylin and eosin (H&E) staining, or positively charged microscope slides for immunohistochemistry and for confocal analysis. Prior to staining, slides were incubated at approximately 60˚C for about one hour for the removal of wax. Ultimately, following staining of the sections, a glass coverslip was mounted onto the tissue using distyrene, a plasticizer, and xylene (DPX) mounting media and left to dry for 48 hours (Dako uorescence mounting medium was used for confocal analysis).
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