All deceased animals were autopsied for macroscopic hints concerning the cause of death. Lung, heart, and liver tissues were taken from selected animals. The tissues were fixed in 4% formalin, dehydrated through graded alcohol series (xylene, 100%, 96% and 70% alcohol and distilled water), and embedded in paraffin wax for histological analysis. Paraffined tissues were cut in sections of 3 μm. Sections were strained with haematoxylin and eosin (Merck KGaA, Darmstadt, Germany, 109249 and 117081) according to the following scheme: 3 minutes covered with haematoxylin, 5 minutes watering in fluent normal water, 1 minute covered with eosin, tapped into water, pulled through an ascending alcohol series and covered with RotiR-Histokitt (Carl Roth GmbH & Co. KG, Karlsruhe, Germany, 6638.1). Subsequently, the histological sections were examined under light microscopy with a LEICA DM 2500 microscope (Leica Microsystems, Nussloch, Germany). Images were captured with a LEICA DFC420 C camera (Leica Microsystems) under bright field illumination at 40x and 100x magnification. The stained sections were used for the descriptive assessment of histological changes.
Haematoxylin
Manufactured by Carl Roth
Sourced in Germany
Haematoxylin is a natural dye extracted from the logwood tree. It is a commonly used staining agent in histology and microscopy to visualize cellular structures and components.
Automatically generated - may contain errors
Lab products found in correlation
Haematoxylin and eosin, by Merck Group (1 mentions)
Leica dm2500 microscope, by Leica Microsystems (1 mentions)
Leica dfc420c camera, by Leica Microsystems (1 mentions)
Xylene, by Carl Roth (1 mentions)
Mirax desk slide scanner, by Zeiss (1 mentions)
Fleximaging v4, by Bruker (1 mentions)
Eosin y, by Merck Group (1 mentions)
Isopropanol, by Merck Group (1 mentions)
Normal goat serum (ngs), by Agilent Technologies (1 mentions)
Agarose, by Carl Roth (1 mentions)
Axiocam mr, by Zeiss (1 mentions)
Sigma fast dab tablets, by Merck Group (1 mentions)
Imager m2, by Zeiss (1 mentions)
Vt1200, by Leica Microsystems (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Vectastain abc kit, by Vector Laboratories (1 mentions)
Axioplan microscope, by Zeiss (1 mentions)
Eclipse e400 microscope, by Nikon (1 mentions)
Click it tunel kit, by Thermo Fisher Scientific (1 mentions)
Entellan, by Merck Group (1 mentions)
7 protocols using haematoxylin
1
Histopathological Examination of Animal Tissues
2
Histological Analysis of Lichen Tissues
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Specimens were immediately fixed in 4% buffered formalin and subsequently embedded in paraffin. Serial histological sections of 4 μm were deparaffinized and rehydrated using graded ethanol and deionized water, respectively. Deparaffined sections were stained with haematoxylin (Carl Roth, Karlsruhe, Germany) and eosin (0.1% in Aqua dest.; Carl Roth) to enable the assessment of the architecture of lichen-affected and healthy tissue.
3
Haematoxylin and Eosin Staining of Tissue Sections
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After acquisition of the mass spectrometry data, the matrix was removed with 70% ethanol, and the tissue sections were stained with haematoxylin and eosin. The sections were transferred to dH2O for 1 min and then haematoxylin (Carl Roth, Karlsruhe, Germany) for 1 min; after washing in tap water for approximately 5 min, they were transferred to eosin Y (Sigma‐Aldrich) for 1 min. The sections were subsequently dehydrated using an increasing alcohol solution series [70%, 90%, and 100% ethanol (Merck) and isopropanol (Merck); 30 s each], transferred to xylene (Carl Roth) for at least 2 min, coverslipped, scanned with a Mirax desk slide scanner (Zeiss, Göttingen, Germany) using a 20× magnification objective, and co‐registered with the respective mass spectrometry imaging data using flexImaging v. 4.0 (Bruker Daltonics).
4
Immunohistochemical Brain Tissue Analysis
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Stainings were performed as described24 . Mice were anesthetized (Ketamine/Xylazine/Heparine) and transcardially perfused with 4% paraformaldehyde (PFA). Brains were dissected and post-fixed in 4% PFA for a minimum of 24 h, transferred to 30% sucrose for cryoprotection for at least 48 h, and embedded in 5% low melting point Agarose (Roth, Germany). 30 µm sagittal brain sections were prepared (VT1200s Leica Microsystems) and, after peroxidase block (1% H2O2 in PBS, 30 min RT), blocked with 10% Normal Goat Serum (Dako, Germany) and 0.1% Triton X-100 (Sigma, Germany)in PBS (1 h, RT), and incubated with primary antibody overnight at 4 °C. After incubation with biotinylated secondary antibodies (1.5 h at RT), proteins were visualized using the ABC method with the Vectastain ABC kit (Vector Laboratories) and the DAB substrate Sigma Fast DAB Tablets (Sigma-Aldrich, Germany). Brain slices were mounted on glass slides and counter-stained with haematoxylin (Roth), followed by dehydration (70%, 80% and 100% ethanol, xylene) and embedding with phenol-free Kaiser’s glycerol gelatine (Roth). Images were taken with a Zeiss AxioCam MR coupled to a Zeiss Axio Imager M2 using the Zeiss Axiovision software.
5
Immunohistochemical Analysis of Primate Tissues
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Immunohistochemistry was carried out as described earlier [30 (link)]. In brief, sections (6 µm) were dewaxed and incubated with the following antibodies: calponin-1 (C-term) (CNN1) monoclonal (1:250; #1806-1, Epitomics, Cambridge, UK), collagen type I (COL1A1) polyclonal (1:1000; OriGene, Rockville, MD, USA), osteoglycin (G-1) (OGN) monoclonal (1:200; sc-374463, Santa Cruz Biotechnology, Inc., Dallas, TX, USA). The nuclei were counterstained with haematoxylin (Carl Roth, Karlsruhe, Germany). Images were taken with a Zeiss Axioplan microscope (Carl Zeiss, Oberkochen, Germany) and digitalized (Jenoptik, Jena, Germany). Per age group sections of at least four monkeys were stained. Controls consisted of omission of the primary antibody.
6
Immunohistochemical Analysis of Tumor Samples
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Tissues were formalin-fixed (CAS 50-00-0) and paraffin-embedded (CAS 8002-74-2), cut into serial tissue sections (5 µm thick), and stained with primary antibodies and their corresponding horseradish peroxidase-linked secondary antibodies (supplementary table E2). For negative controls, primary antibodies were omitted. TUNEL was performed with the Click-iT TUNEL kit (Thermo Fisher). For negative controls, dUTP (CAS 94736-09) was omitted. Slides were counterstained with haematoxylin (Roth, Karlsruhe, Germany; CAS 517-28-2) and coverslipped using Entellan (Merck, Darmstadt, Germany). 10 different areas of each tumour and five different fields of view of each tissue section were analysed by three trained blinded readers (A-S.L., W.K. and G.T.S.) at low magnification (×20), the percentage of stained cells was semiquantitatively scored as 0 (<5%), 1 (5-24%), 2 (25-49%), 3 (50-74%) or 4 (>74%) on an Eclipse E400 microscope (Nikon, Melville, NY, USA; RRID:SCR_020320) using TCapture software (Tucsen Photonics, Fuzhou, China; RRID:SCR_020956) and the results were averaged by patient, as routinely done and described elsewhere [17] [18] [19] [20] 23] . Cancer-specific hallmark expression was also determined in randomly selected paired normal lung tissues (n=50).
7
Histochemical Analysis of Liver and Intestine
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Frozen liver sections (10 µm) were stained with Oil red O (Sigma-Aldrich), as described previously (17) . Liver sections (5 µm) embedded in paraffin were stained with haematoxylineosin (Sigma-Aldrich) to evaluate the histologic features as detailed before (14) . To determine goblet cell number being indicative of mucous formation (18) , duodenal sections (4 µm) embedded in paraffin were consecutively stained with Alcian blue (30 min), periodic acid (10 min), Schiff's reagent (15 min) (all from Carl-Roth) and counterstained with haematoxylin. Quantitative evaluation of staining was carried out as previously described in detail (19) . Representative photomicrographs of Oil red O (100×) and haematoxylin-eosin (200×), as well as goblet cell (200×) staining, were captured using the system incorporated in the microscope (Leica DM4000 B LED).
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