Mouse tissue samples were resected, formalin‐fixed and paraffin‐embedded. Four‐micrometre tissue sections were prepared, deparaffinised and rehydrated for further staining. For IHC staining, the EDTA antigen retrieval solution and UltraSensitive™ SP (Mouse/Rabbit) IHC Kit (Maxim Biotechnologies, Fuzhou, China) were used according to the instructions provided by the manufacturer. Primary antibodies against human Ki67, human EGFR (both from Maxim Biotechnologies) or human CD8 (Abcam) were incubated at 4 °C overnight at an optimised concentration. Stained tissue sections were developed with the Diaminobenzidine (DAB) Kit (Maxim Biotechnologies) for 1 min and counterstained with haematoxylin solution (Sigma‐Aldrich) for 10 min. For H&E staining, sections were stained with haematoxylin solution (Sigma‐Aldrich) for 5 min and eosin Y solution (Sigma‐Aldrich) for 1 min. For TUNEL assays, sections were treated with 20 μg mL−1 proteinase K (AMRESCO, Solon) at 37 °C for 20 min and then washed in PBS. The commercially available Colorimetric TUNEL Apoptosis Assay Kit (Beyotime Institute of Biotechnology, Shanghai, China) was used to detect apoptotic cells according to the instructions provided by the manufacturer. The histopathological images were obtained and analysed using the Cellsens Standard software (Olympus).
Haematoxylin solution
Manufactured by Merck Group
Sourced in United States
Haematoxylin solution is a laboratory reagent used in histology and cytology. It is a staining solution that is commonly used to stain cell nuclei blue in various microscopy techniques.
Automatically generated - may contain errors
Lab products found in correlation
3 3 diaminobenzidine (dab), by Agilent Technologies (2 mentions)
Eosin solution, by Merck Group (2 mentions)
9 aminoacridine 9 aa, by Merck Group (1 mentions)
Dpx mounting media, by Merck Group (1 mentions)
Indium tin oxide glass slides, by Bruker (1 mentions)
Gill no 2, by Merck Group (1 mentions)
Milli q system, by Merck Group (1 mentions)
Sodium chloride (nacl), by Merck Group (1 mentions)
Acetonitrile, by Merck Group (1 mentions)
Methanol, by Merck Group (1 mentions)
Ethanol, by Merck Group (1 mentions)
α cyano 4 hydroxycinnamic acid, by Bruker (1 mentions)
Human cd8, by Abcam (1 mentions)
Proteinase k, by Avantor (1 mentions)
Colorimetric tunel apoptosis assay kit, by Beyotime (1 mentions)
Eosin y solution, by Merck Group (1 mentions)
Cellsens standard software, by Olympus (1 mentions)
Mounting solution, by Merck Group (1 mentions)
Microscope slide, by Thermo Fisher Scientific (1 mentions)
Leica application suite x, by Leica Microsystems (1 mentions)
15 protocols using haematoxylin solution
1
Immunohistochemistry and TUNEL Assay for Mouse Tissues
2
Immunohistochemical Analysis of JQ1 Treatment
3
Mass Spectrometry-Based Tissue Imaging
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The isoflurane anaesthetic and nitrogen 4.0 were obtained from Baxter and Linde (both Bratislava, Slovak Republic), respectively. The indium-tin oxide (ITO) glass slides and α-cyano-4-hydroxycinnamic acid (CHCA) were obtained from Bruker Daltonics (Bremen, Germany). Acetonitrile, methanol, ethanol and water (all LC-MS grade) were obtained from Merck Millipore (Prague, Czech Republic). Trifluoroacetic acid (TFA), chloroform, sodium chloride, 9-aminoacridine (9-AA), haematoxylin solution, Gill no. 2 and DPX mounting media were obtained from Sigma-Aldrich (Prague, Czech Republic). Eosin Y (0.5%) aqueous solution was purchased from VWR Chemicals (Stribrna Skalice, Czech Republic). Hydrochloric and nitric acid (65%, Analpure) were obtained from FlukaBioChemika or Analytika Ltd., respectively (both Prague, Czech Republic). Deionized water (18.2 MΩ/cm) was prepared using a Milli-Q system (Millipore, Molsheim, France). Omni Slides with hydrophobic surfaces were purchased from Prosolia, Inc. (Zionsville, USA).
4
Histological Analysis of Mouse Skin
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Mouse skin separated from back was embedded with OCT solution in frozen section. Frozen section was cut into 6‐μm‐thick sections under a microscope (Cryostat CM1850, Leica Biosystems, ) and then attached to microscope slides (Thermo Fisher Scientific,). For histological analysis, sections were stained 3% haematoxylin solution (Sigma) for 5 min and then rinsed in cool running ddH2O for 5 min. Sections were dipped in 0.5% eosin (Sigma) solution and then rinsed in cool running ddH2O for 5 min. Subsequently, sections were dipped in 50%, 70% and 95% ethanol and xylene. After sections were covered coverslip with mounting solution (Sigma). The thickness of the epidermis and the COX‐2 were visualized using a fluorescent microscope (Leica microsystems,) and images were analysed using Leica Application Suite X (Leica microsystems) software.
5
Hematoxylin Staining of Heat-Shocked Cells
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For hematoxylin staining, heat-shocked cells were washed once with PBS and fixed in 3:1 methanol-acetic acid for 10 min. Slides were air dried and aged overnight in an oven at 37 °C. Cells were stained with haematoxylin solution (Sigma-Aldrich) for 8 min, washed in distillate water and air dried.
6
Haematoxylin-Eosin Staining and Cell Counting
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Cells were fixed with 4% paraformaldehyde in PBS for 15 min and plunged in PBS and then in Haematoxylin solution (Sigma) for 20 min. Haematoxylin staining was stopped by washing in distilled water and followed by plunging cells in the eosin solution (Sigma) for a few min. After repeated washing to remove the excess of dye, cells were dehydrated in increasing alcohol solutions, clarified in xylene, and finally covered with the DPX mounting medium (Sigma). Cell count was performed at light microscopy at 40x magnification. Briefly, for each experimental group, the number of stained cells detectable after each specific treatment was counted and expressed as a percentage of the control group. These values represent the means of six independent cell counts.
Moreover, we counted the number of giant cells occasionally observed after 10 μM METH. We considered as giant cells those owning a diameter higher than 14–15 μm. The amount of giant cells out of the total number of cells counted on the glass slide was expressed as a percentage, for each experimental group. The values represent the means of six independent cell counts.
Moreover, we counted the number of giant cells occasionally observed after 10 μM METH. We considered as giant cells those owning a diameter higher than 14–15 μm. The amount of giant cells out of the total number of cells counted on the glass slide was expressed as a percentage, for each experimental group. The values represent the means of six independent cell counts.
7
Histological Staining of Lung Tissue
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Paraffin-embedded lung tissue slices were dewaxed and hydrated. The nuclei were stained with haematoxylin solution (Sigma Aldrich). Following this, cytoplasmic staining was performed using an eosin staining solution (Sigma Aldrich). After the sections were dried, the sheets were preserved using neutral resin.
8
Histological Analysis of Aortic Arch
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The aortic arch to the branch of the abdominal aorta was dissected and immersed in precooled 4% paraformaldehyde (PFA, Sigma‐Aldrich) in PBS at 4°C overnight. After a brief wash with 1 × phosphate‐buffered saline, the aortic arch was processed directly for paraffin embedding and microtome sectioned at thickness 5 nm by a microtome (HistoCore BIOCUT, Leica, USA) according to the previous method.21 After serial dewax and rehydration, the sections were stained by haematoxylin solution (Sigma‐Aldrich) for 5–10 min. After being rinsed under tap water, the tissues were differentiated using 1% aqueous hydrochloric acid, and the development of staining was checked under a microscope. After rinsing for 5 min in running tap water, the sections were counterstained by eosin alcoholic solution for 0.5–1 min. Finally, the sections were dehydrated by increasing serial concentration of ethanol (70%, 90%, and 100% ethanol, each for 2 min) and cleared in xylene for 2 min. After mounting with neutral resin, the sections were imaged under a microscope with a camera. Two independent pathologies evaluated the lesions in a blinded manner.
9
Immunohistochemical Analysis of Spermatogenesis
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The formalin-fixed, paraffin-embedded sections presenting normal spermatogenesis were deparaffinized in xylene (2 × 10 min) and rehydrated 2 × 5 min in 100% ethanol, 96% ethanol, and 70% ethanol. Antigen retrieval was performed as follows: incubation in 2% NaBH4 for 30 min at room temperature, incubation for 30 min in 0.1% glycine at room temperature, and incubation in 0.01% sodium citrate solution at 95 °C for the next 30 min. Afterwards, the endogenous peroxidase was blocked with 3% H2O2 (in PBS) for 30 min. After rinsing with PBS, the histological section was blocked with 10% goat serum (in PBS) for 1 h and then incubated overnight with primary antibody (diluted in 1xPBS) at 4 °C. After rinsing 2 × 5 min with PBS, the section was incubated with secondary antibody (diluted in PBS) for 1 h at room temperature. Finally, the preparation was incubated with 4 drops of AEC Chromogen Kit (Sigma-Aldrich, St. Louis, MO, USA) for 10 min; after rinsing with H2O, the haematoxylin solution (Sigma-Aldrich, St. Louis, MO, USA) was applied for 1 min. The antibody dilutions are listed in Table S4 .
10
Cell Migration and Colony Formation Assay
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After RP11-366H4.1.1, LINC460 or AC093850.2 was subjected to individual knockdown, KYSE150 or KYSE70 cell migration and colony formation assays were performed as previously described [16 (link)]. Briefly, at 24 h post transfection, cells were starved for 12 h with serum-free medium (Invitrogen, California, USA) and then 5 × 104 cells were plated in serum-free medium in the upper well of a transwell chamber (24-well insert; pore size, 8 μm; BD Biosciences, Franklin Lakes, NJ, USA), and the lower chamber containing medium with 10% FBS. After 48 h, cells in the top chamber were removed with a cotton swab and only cells that migrated through the pores were fixed and stained in haematoxylin solution (Sigma-Aldrich, St. Louis, MO, USA) and counted. For colony formation, 500 cells per well in 24-well plate were incubated in medium supplemented with 10% FBS for ten days, and then colonies were stained with haematoxylin solution and observed.
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