Prior to staining, sections incubated for 30 min at 60 °C followed by incubation for 20 min in xylene (9713.2, Carl Roth, Karlsruhe, Germany). After hydrogenation by a descending alcohol series, they were stained with Mayer hematoxylin solution (1.07961.0500, Sigma Aldrich, St. Louis, MO, USA) for 10 min. After 5 min under running warm water, sections were counterstained for one minute with 0.5% eosin G solution (X883.2, Carl Roth, Karlsruhe, Germany). Again, they were rinsed with warm water and dehydrated by the ascending series of alcohol. After 20 min in xylene, they were covered using entellan (1.07961.0500, Merck, Darmstadt, Germany). The stained histological sections were digitized using an Olympus IX50 microscope (Hamburg, Germany) at 100 fold magnification, and evaluation of root resorptions at the disto-buccal tooth root of the moved first molar in relation to the respective root surface area was assessed with ImageJ (Ver.1.4.7, National Institutes of Health, Bethesda, MD, USA), as described before [16 (link),19 (link)].
Mayer hematoxylin solution
Manufactured by Merck Group
Sourced in Germany
Mayer's hematoxylin solution is a laboratory reagent used for staining and visualization of cellular structures in microscopy. It is a commonly used staining solution in histology and cytology. The solution contains hematoxylin, a natural dye that binds to the nucleic acids in cells, allowing the visualization of cell nuclei.
Automatically generated - may contain errors
Lab products found in correlation
Goat anti rabbit igg, by Agilent Technologies (3 mentions)
Dpx mounting medium, by Merck Group (2 mentions)
Eosin y solution, by Merck Group (2 mentions)
Ix50 microscope, by Olympus (2 mentions)
Entellan, by Merck Group (2 mentions)
Horseradish peroxidase conjugated rabbit anti mouse igg, by Agilent Technologies (2 mentions)
Setdb1, by Santa Cruz Biotechnology (1 mentions)
H3k9me3, by Merck Group (1 mentions)
Horseradish peroxidase conjugated goat anti rabbit igg, by Agilent Technologies (1 mentions)
Caspase 3 primary antibody, by Cell Signaling Technology (1 mentions)
9 protocols using mayer hematoxylin solution
1
Histological Evaluation of Tooth Root Resorption
2
Tissue Histological Staining Protocol
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10 μm sections obtained from paraffin embedded tissue were processed in the following steps in order: 2 × 10-min deparaffinization with xylene; 2 × 5-min rehydration in 100% ethanol; 2 min 95% ethanol and 2 min with 70% ethanol; wash in distilled water; 8 min stain in Mayer hematoxylin solution (Sigma-Aldrich, MHS1); 10 min rinse in warm running tap water; wash in distilled water for 30 s; dip slides in 95% ethanol 10 times; stain in Eosin Y solution (Sigma-Aldrich, HT110116) for 30 s; 95% ethanol 5 min; 100% ethanol 5 min each 2 times; clear in xylene 5 min each 2 times; mount with DPX Mounting medium (Millipore Sigma, 06522).
3
Lung Tissue Preparation and H&E Staining
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Sectioning: The excised lungs were left in 10% neutral-buffered formalin (NBF) overnight and then immersed in a solution of 20% sucrose in 1X PBS. The lungs were then placed in optimal cutting temperature compound (OCT) (TFMTM, General Data Company Inc., Cincinnati, OH, USA). Sectioning was performed using a Leica CM 1860 cryostat (Leica Byosystems, Deer Park, IL, USA) by cutting 8 μm sections of the left and right lung of mice injected with WT SUM-149 or MTDH-edited cells (n = 7/group). The sections were mounted on slides with a subbing solution as described in [78 (link)].
Hematoxylin & Eosin (H & E) staining: First, tissues were stained with Mayer hematoxylin solution (Sigma-Aldrich) for 10 min at RT and then rinsed with running tap water for 10 min until the water was colorless. The tissues were then rinsed in 95% alcohol for 10 s. Then, the staining was performed with Eosin Y with Phloxine B (Sigma-Aldrich) solution for 30 s and rinsed in ascending series of ethanol (70%, 95%, and 100%). Finally, tissues were processed in xylene 1 min twice and mounted using VectaMount® permanent mounting medium (Vector Labortories, Newark, CA, USA). A pathologist analyzed H&E-stained slides and the micrographs were captured at a magnification of 20×.
Hematoxylin & Eosin (H & E) staining: First, tissues were stained with Mayer hematoxylin solution (Sigma-Aldrich) for 10 min at RT and then rinsed with running tap water for 10 min until the water was colorless. The tissues were then rinsed in 95% alcohol for 10 s. Then, the staining was performed with Eosin Y with Phloxine B (Sigma-Aldrich) solution for 30 s and rinsed in ascending series of ethanol (70%, 95%, and 100%). Finally, tissues were processed in xylene 1 min twice and mounted using VectaMount® permanent mounting medium (Vector Labortories, Newark, CA, USA). A pathologist analyzed H&E-stained slides and the micrographs were captured at a magnification of 20×.
4
Tissue Histological Staining Protocol
5
Immunohistochemical Analysis of SETDB1 and H3K9me3
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The 6-μm-thick sections were de-paraffinized with xylene and re-hydrated with ethanol series from 100% (v/v) to 70% (v/v). The slides were washed three times, each for 5 min in PBS, followed by 0.5% Triton X-100 in ultrapure water for 10 min at room temperature. The slides were washed and boiled in a solution of 0.01-M Tris-EDTA (pH = 9) for 15 min for antigen retrieval, and cooled at room temperature for 40 min. The sections were incubated with 3% H 2 O 2 to block endogenous peroxidase, washed three times with PBS, and blocked with 10% goat serum for 2 h at room temperature. Subsequently, the slides were incubated with the primary antibodies against SETDB1 (1:50; Santa Cruz Biotechnology), or H3K9me3 (1:50; Millipore) at 4°C overnight. While some slides were incubated with blocking solution as a negative control. Next day, the slides were rinsed four times in PBS, incubated with biotinylated secondary antibody for 1 h at 37°C and then washed three times with PBS. The slides were exposed to the horse radish peroxidase at 37°C for 1 h, followed by incubation in 3,3′-diaminobenzidine (CWBIO, Beijing, China) for staining and counterstained with Mayer hematoxylin solution (Sigma-Aldrich), and washed three times with PBS for 5 min each. Digital images were captured with the Nikon Eclipse 80i microscope camera.
6
Immunohistochemical Quantification of PMN
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To detect polymorphonuclear cells (PMN) in kidney and liver, immunohistochemistry was performed on 5 μm tissue cryosections. Sections were fixated for 10 min using acetone. Next, sections were stained with HIS-48 mAb (supernatant, two times diluted) using an indirect immunoperoxidase technique. Endogenous peroxidase was blocked using H2O2 0.01% in phosphate-buffered saline for 30 min. After thorough washing, sections were incubated with horseradish peroxidase-conjugated rabbit anti-mouse IgG as a secondary antibody for 30 min, followed by goat anti-rabbit IgG as a tertiary antibody for 30 min (both from Dako, Glostrup, Denmark). The reaction was developed using 9-amino-ethylcarbazole as chromogen and H2O2 as substrate. Sections were counterstained using Mayer hematoxylin solution (Merck, Darmstadt, Germany). Negative antibody controls were performed. Localization of immunohistochemical staining was assessed by light microscopy. For each tissue sample, positive cells were counted in 10 microscopic random fields of the tissue at 40x magnification. Results were presented as number of positive cells per glomerulus in the kidney and number of positive cells per area (μm2) in the liver.
7
Detecting Polymorphonuclear Cells in Kidney and Liver
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To detect polymorphonuclear cells in kidney and liver, immunohistochemistry was performed on 5- μm tissue cryosections. Sections were fixated for 10 min using acetone. Next, sections were stained with HIS-48 mAb (supernatant, two times diluted) using an indirect immunoperoxidase technique. Endogenous peroxidase was blocked using H 2O2 0.01% in phosphate-buffered saline for 30 mins. After thorough washing, sections were incubated with horseradish peroxidase-conjugated rabbit anti-mouse IgG as a secondary antibody for 30 mins, followed by goat anti-rabbit IgG as a tertiary antibody for 30 mins (both from Dako, Glostrup, Denmark). The reaction was developed using 9-amino-ethylcarbazole as chromogen and H 2O2 as substrate. Sections were counterstained using Mayer hematoxylin solution (Merck, Darmstadt, Germany). Negative antibody controls were performed. Localization of immunohistochemical staining was assessed by light microscopy. For each tissue section, positive cells per field were counted in 10 microscopic fields of the tissue at 40x magnification. Results were presented as number of positive cells per glomerulus in the kidney and number of positive cells per field in the liver.
8
Histological Analysis of Root Resorption
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Sections were deparaffinized at 60 °C for 30 min and transferred to xylene (9713.2, Carl Roth GmbH, Karlsruhe, Germany) for 20 min. They were hydrogenated by a descending alcohol series followed by staining with Mayer hematoxylin solution (1.07961.0500, Merck KG, Darmstadt, Germany) for 10 min. After incubation under running warm water for 5 min, slides were counterstained with eosin G solution 0.5% (X883.2, Carl Roth GmbH, Karlsruhe, Germany) for one minute. After rinsing the samples under warm tap water, they were dehydrated by an ascending series of alcohol. After incubation for 20 min in xylene, the coverslips were applied with entellan (1.07961.0500, Merck KGaA, Darmstadt, Germany). The stained histological sections (Figure 2b) were digitized under the microscope (Olympus IX50 microscope in combination with DP2-SAL camera, Olympus, Hamburg, Germany) and evaluation of the relative extent of root resorption area at the distobuccal root of the upper first molar in relation to the respective total root area within the same slice was performed with ImageJ (Ver.147, National Institutes of Health, Bethesda, MD, USA), as described in Kirschneck et al. [25 (link),27 (link),38 (link)].
9
Immunohistochemical Detection of Caspase-3 in Liver and Kidney
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To detect caspase-3 positive cells in liver and kidney, immunohistochemistry was performed on 3 μm sections of paraffin embedded liver samples. Sections were deparaffined in a sequence of xylene, alcohol and water. As an antigen retrieval method we used for caspase-3 samples: EDTA (1 mM, pH 8.0) buffer. Next, sections were stained with Caspase-3 primary Antibody (Cell Signaling cat. nr. 9661, 100× diluted in 1 % BSA/PBS) using an indirect immunoperoxidase technique. Endogenous peroxidase was blocked using H2O2 0.3 % in phosphate-buffered saline for 30 min. After thorough washing, sections were incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG as a secondary antibody for 30 min (Dako, Glostrup, Denmark. cat. nr. P0448), followed by rabbit anti-goat IgG as a tertiary antibody for 30 min (Dako, Glostrup, Denmark. cat. nr. P0449). The reaction was developed using DAB as chromogen and H2O2 as substrate. Sections were counterstained using Mayer hematoxylin solution (Merck, Darmstadt, Germany). Negative antibody controls were performed. Localization of immunohistochemical staining was assessed by light microscopy. For each tissue section, positive cells per field were counted by a blinded researcher in ten microscopic fields of the tissue at 10× magnification. Results were presented as number of positive cells per field.
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