After adipogenic differentiation, the media was harvested, and the cells fixed with 4% PFA (Sigma-Aldrich/Merck) as described above. Then, cells were incubated with 60% isopropanol for 5 min at RT. Cells were stained with Oil Red O for 5 min in RT and washed twice with PBS. Cells were stained with haemotoxylin (H9627, Sigma-Aldrich/Merck) for 1 min at RT. Cells were observed using an inverted microscope (Leica, Germany).
Haemotoxylin
Manufactured by Merck Group
Sourced in United States, Germany
Haemotoxylin is a staining dye commonly used in histology and pathology for the visualization of cell nuclei. It is a natural plant-derived compound that binds to the DNA and RNA within cell nuclei, providing a distinct blue-purple coloration. Haemotoxylin is a core component of many standard staining procedures in microscopy and laboratory analysis, enabling the clear identification and differentiation of cellular structures.
Automatically generated - may contain errors
Lab products found in correlation
Inverted microscope, by Leica (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Toluidine blue, by Merck Group (1 mentions)
Stempro osteogenesis differentiation kit, by Thermo Fisher Scientific (1 mentions)
Neutral buffered formalin, by Merck Group (1 mentions)
Chondrogenesis differentiation kit, by Thermo Fisher Scientific (1 mentions)
Adipogenesis differentiation kit, by Thermo Fisher Scientific (1 mentions)
Oil red o, by Merck Group (1 mentions)
Alizarin red s, by Merck Group (1 mentions)
24 well plate, by Corning (1 mentions)
Normal goat serum (ngs), by Vector Laboratories (1 mentions)
Vector abc reagent, by Vector Laboratories (1 mentions)
Imagescope software, by Leica (1 mentions)
Paraffin wax, by Merck Group (1 mentions)
Doxorubicin, by Merck Group (1 mentions)
Formalin, by Merck Group (1 mentions)
α lipoic acid, by Merck Group (1 mentions)
Ethanol, by Merck Group (1 mentions)
Sodium hydroxide (naoh), by Merck Group (1 mentions)
Sodium chloride (nacl), by Merck Group (1 mentions)
6 protocols using haemotoxylin
1
Adipogenic Differentiation and Oil Red O Staining
2
Multilineage Differentiation of Mesenchymal Stem Cells
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Cells (ovAD-MSCs, passage 3) were split and seeded onto 24-well plates (Corning) in DMEM2 at a concentration of 100,000 cells/mL (0.5 mL) and incubated until monolayers were 80% confluent. Media was changed to osteogenic, chorondrogenic or adipogenic differentiating medias (1 mL) (StemPro™ Osteogenesis Differentiation Kit, Adipogenesis Differentiation Kit, Chondrogenesis Differentiation Kit, Life technologies, Carlsbad, US). Cells were maintained for two weeks in the respective media, with media changed every 3 days. Differentiated cell monolayers were rinsed in DPBS (1 mL) then fixed in 10% neutral buffered formalin (1 mL) (Sigma-Aldrich) for 10 mins at rt°C. Monolayers of adipocytes were stained with Oil Red O (0.5% w/v isopropanol, 1 mL, rt°C, 10 min) (Sigma-Aldrich) and counter stained with 0.1% haemotoxylin (1 mL, rt°C, 10 min) (Sigma-Aldrich). Osteocytes were stained with 2% Alizarin Red S (1 mL, rt°C, 10 min) (Sigma-Aldrich). Chondrocytes were stained with Toluidine Blue (1 mL, 0.1% w/v, rt°C, 10 min) (Sigma-Aldrich). Stained monolayers were rinsed with ROH2O (3x, 1 mL) then imaged using an Olympus inverted phase contrast and microscope (IX51, Olympus, Tokyo, Japan).
3
Immunohistochemical Analysis of MUC1 in Prostate Cancer
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IHC was performed on paraffin embedded and serially cut prostate cancer tissues obtained from St. Joseph's Hospital, Hamilton, Ontario, Canada. Slides were deparaffinized in xylene and cleared in an ethanol series. Antigen retrieval was performed in a food steamer for 20 minutes using sodium citrate buffer (pH = 6.0). Tissues were blocked for 1 hour in PBS containing 1% BSA and 10% normal goat serum (Vector Laboratories). MUC1-N (1:200, BD) and MUC1-C (1:10, Fishr Scientific) antibodies were incubated overnight at 4°C. Secondary antibody biotinylated goat anti-mouse IgG or anti-hamster IgG, respectively, and Vector ABC reagent (Vector Laboratories) were incubated according to the manufacturer's instructions. Secondary antibody only was used as negative control. Washes were performed with PBS. Chromogenic reaction was carried out with diaminobenzidine (Vector Laboratories), and slides were counterstained with haemotoxylin (Sigma Aldrich). Image analysis was performed using ImageScope software (Leica Microsystems Inc.). Staining intensity values derived from ImageScope were converted to an HScore using the formula [HScore = (% Positive) x (intensity) + 1]. The HScore was normalized through background subtraction and averaged amongst ≤ 5 images per tissue sample.
4
Histochemical Analysis of Tissue Samples
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Sodium hydroxide, sodium chloride, doxorubicin, α-lipoic acid, formalin, potassium dihydrogen phosphate and dipotassium hydrogen phosphate were purchased from Sigma Chemical Co., Saint Louis, MO, USA. Diethylether, ethanol, xylene, paraffin wax, haemotoxylin and eosin were purchased from Sigma Chemical Co., (St Louis, Mo USA). All other chemicals were supplied by Zayo Company, Jos, Nigeria, which is an accredited supplier of Sigma and BDH chemicals in Nigeria. All reagents and chemicals used were of analytical grade (greater than or equal to 99.7%).
5
Visualizing Immune Cells in Mouse Brains
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Whole brain from heparin-PBS perfused mice were embedded in OCT freezing medium over liquid nitrogen. The brain stem and cerebellum were sectioned by cryostat (10 μm thickness) onto slides coated with APS (3-Aminopropyltriethoxy-silane) and stained for FA11-CD68 and KT3-CD3 using the avidin–biotin–peroxidase complex method (ABC, Thermo Scientific); 0.015% (v/v) hydrogen peroxide as substrate and diaminobenzidine (Sigma) as chromagen and counterstained with haemotoxylin (Sigma). Slides were dewaxed in Histoclear I and II (ABC Scientific) and coverslips were mounted with DPX mountant (Sigma). All pictures were taken using Cell-1 software (Leica).
6
Ovarian Histology in Constant Dark
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Age-matched cohorts of 13–26-week-old female mice were singly-housed and placed in light-tight chambers for 8 weeks to detect the effect of constant darkness conditions on ovarian function. A subset of mice had locomotor behavior monitored by passive infrared motion detectors to detect locomotor activity (Coral Plus, Visonic, Bloomfield, CT) or Mini Mitters as described above. Cages were changed every 3-4 weeks in red light. Mice were sacrificed in the morning without coordination with activity cycle because the relative histological features are long-lasting. Ovaries were dissected and placed in a mixture of 60% EtOH, 30% formaldehyde (40%), and 10% acetic acid for 24 hours, followed by immersion in 70% EtOH. Ovaries were paraffin processed by Reveal Biosciences (San Diego, CA), embedded, and sectioned at 10 µm. Haemotoxylin (Sigma-Aldrich) and eosin (Leica, Wetzlar Germany) staining was performed and the sections were imaged using an Olympus V200 Slide Scanner (UCSD Neurosciences Microscopy Core). Ovarian structures were quantified by two blinded observers and averaged, and the greatest number of structures per section for each animal was reported to avoid double-counting. Graafian follicles were determined based on the presence of an antral space.
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