Sections of formalin-fixed and paraffin-embedded skin samples were sectioned on a microtome at 3 µm, deparaffinised in xylene or Roticlear (Roth), and rehydrated through a graded series of alcohols to deionised water before staining. For epidermal thickness determination, slides underwent staining by haematoxylin (Sigma) and eosin G (Roth). Image capture was conducted using an Axioplan imaging fluorescence microscope (Zeiss). Epidermal measurements (distance from the top of the living epidermis to the end of the rete ridge) were done using microscope-associated AxioVision software version 4.6.3. For CD3 staining, slides underwent heat-induced epitope retrieval in a citrated buffer (pH 6) at 110 °C for 20 min, followed by peroxidase block in Dako REAL peroxidase-blocking solution (DAKO) for 10 min. CD3 was detected using polyclonal rabbit anti-human CD3 antibody (DAKO; 1:100) and visualised using DAKO REAL Detection System (AP/RED, rabbit/mouse) (DAKO). haematoxylin (Mayer’s haemalaun; Sigma) solution was used for tissue counterstaining. After image capture using a BZ-X800 Fluorescence microscope (Keyence), cell counting was performed using Fiji ImageJ software v2.9.0.
Haematoxylin
Manufactured by Merck Group
Sourced in United States, Germany, United Kingdom, India, Ireland, China, Canada, Australia
Haematoxylin is a natural dye extracted from the heartwood of the Logwood tree. It is a common staining agent used in histology and cytology laboratories for the visualization of cellular structures.
Automatically generated - may contain errors
Lab products found in correlation
Safranin o, by Merck Group (7 mentions)
3 3 diaminobenzidine (dab), by Merck Group (6 mentions)
Oil red o, by Merck Group (6 mentions)
Eosin y, by Merck Group (5 mentions)
Dibutylphthalate polystyrene xylene (dpx), by Merck Group (5 mentions)
Ethanol, by Merck Group (5 mentions)
Vectastain universal elite abc kit, by Vector Laboratories (4 mentions)
Paraformaldehyde (pfa), by Merck Group (4 mentions)
Fast green, by Merck Group (4 mentions)
Bovine serum albumin (bsa), by Merck Group (4 mentions)
Sodium hydroxide (naoh), by Merck Group (4 mentions)
Xylene, by Merck Group (4 mentions)
Eosin solution, by Merck Group (4 mentions)
Horseradish peroxidase streptavidin detection system, by Agilent Technologies (3 mentions)
Lsm 510, by Zeiss (3 mentions)
Dpx mounting medium, by Merck Group (3 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (3 mentions)
Formalin, by Merck Group (3 mentions)
Iscript cdna synthesis kit, by Bio-Rad (3 mentions)
Trizol, by Thermo Fisher Scientific (3 mentions)
217 protocols using haematoxylin
1
Epidermal Thickness and CD3 Immunostaining
2
Preparation of Mayer's Haematoxylin and Eosin Stains
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See the Supplementary Information for information on all other reagents and solutions.
Mayer's haematoxylin solution was prepared by dissolving 5 g of aluminium potassium sulphate dodecahydrate (Merck Millipore, cat. 1010421000) in 100 mL of water, and adding 1 g of haematoxylin (Merck, cat. H9627). After complete dissolution, 0.02 g of sodium iodide (Merck, cat. 1065230100) was added and completely dissolved. Then, 2 mL of acetic acid (Sigma-Aldrich, cat. 33209) was added, and then the solution was boiled and then cooled. Once ready to use, the solution was filtered using a 0.45 m filter. Eosin (5%) solution was prepared by dissolving 0.5 g of Eosin Y (Sigma-Aldrich, cat. 230251) in 99 mL water/1 mL acetic acid.
preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
Mayer's haematoxylin solution was prepared by dissolving 5 g of aluminium potassium sulphate dodecahydrate (Merck Millipore, cat. 1010421000) in 100 mL of water, and adding 1 g of haematoxylin (Merck, cat. H9627). After complete dissolution, 0.02 g of sodium iodide (Merck, cat. 1065230100) was added and completely dissolved. Then, 2 mL of acetic acid (Sigma-Aldrich, cat. 33209) was added, and then the solution was boiled and then cooled. Once ready to use, the solution was filtered using a 0.45 m filter. Eosin (5%) solution was prepared by dissolving 0.5 g of Eosin Y (Sigma-Aldrich, cat. 230251) in 99 mL water/1 mL acetic acid.
preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
3
Histological Evaluation of Rat Ileum and Colon
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Biopsies were taken from the ileum and colon and then fixed in 10% neutral‐buffered formalin for a week at room temperature. Paraffin sections with a thickness of 4–5 μm were prepared using a rotary microtome (Leica Biosystems). The sections obtained were stained with haematoxylin and eosin stain (haematoxylin: Cas No: 517‐28‐2; Eosin: Cas No: 17372‐87‐1, Merck, Germany) to visualize the general architecture of the rat ileum and colon. Random evaluation of approximately 10–15 areas per animal section was performed to observe histological alterations.20 Histopathological changes were evaluated under a light microscope by two blinded researchers.
4
Hematoxylin and Eosin Staining Procedure
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See the Supplementary Information for information on all other reagents and solutions.
Mayer's haematoxylin solution was prepared by dissolving 5 g of aluminium potassium sulphate dodecahydrate (Merck Millipore, cat. 1010421000) in 100 mL of water, and adding 1 g of haematoxylin (Merck, cat. H9627). After complete dissolution, 0.02 g of sodium iodide (Merck, cat. 1065230100) was added and completely dissolved. Then, 2 mL of acetic acid (Sigma-Aldrich, cat. 33209) was added, and then the solution was boiled and then cooled. Once ready to use, the solution was filtered using a 0.45 µm filter. Eosin (5%) solution was prepared by dissolving 0.5 g of Eosin Y (Sigma-Aldrich, cat. 230251) in 99 mL water/1 mL acetic acid.
Mayer's haematoxylin solution was prepared by dissolving 5 g of aluminium potassium sulphate dodecahydrate (Merck Millipore, cat. 1010421000) in 100 mL of water, and adding 1 g of haematoxylin (Merck, cat. H9627). After complete dissolution, 0.02 g of sodium iodide (Merck, cat. 1065230100) was added and completely dissolved. Then, 2 mL of acetic acid (Sigma-Aldrich, cat. 33209) was added, and then the solution was boiled and then cooled. Once ready to use, the solution was filtered using a 0.45 µm filter. Eosin (5%) solution was prepared by dissolving 0.5 g of Eosin Y (Sigma-Aldrich, cat. 230251) in 99 mL water/1 mL acetic acid.
5
Visualization of Tissue Composition
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Sections of native lung tissue and decellularized matrices were stained to visualize total collagen using a Masson’s trichrome stain kit (Sigma-Aldrich) according to the instructions of the manufacturer. Sections of recellularized matrices were stained with haematoxylin and eosin staining through consecutive incubation in the following solutions at room temperature: haematoxylin (Merck, Kenilworth, United States) for 5 min, regular tap water for 5 min, eosin (Chroma, Bellows Falls, United States) for 2 min, washed twice in 96% ethanol and twice in 100% ethanol. After staining, the dehydrated slides were mounted in Tissue-Tek Coverslipping Film with the Sakura Tissue-Tek Film Coverslipper.
6
Quantifying Spheroid Cell Proliferation and Apoptosis
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Cells were harvested carefully to ensure they retained their spheroid structure, pellets were resuspended in HistoGel (Thermo Scientific, Waltham, Massachusetts, US), fixed with 4% PFA (VWR International, Radnor, Pennsylvania, US), and embedded in paraffin. Sections of 4 μm were stained with haematoxylin (Mayer’s, Sigma-Aldrich Chemie, St. Louis, Missouri, US) and eosin (Merck Chemicals GmbH, Darmstadt, Germany) or Ki-67 (Clone SP6, monoclonal rabbit IgG, Order-no. 275R-16, LOT-no. 0000075544, Cell Marque, Rocklin, California, US). After heat-induced antigen retrieval at pH 9, anti-Ki-67 (1:100) was incubated for 30 minutes. For quantification of apoptosis, TUNEL assay (ApopTag® Plus Peroxidase In Situ Apoptosis Kit, Merck Chemicals GmbH, Darmstadt, Germany) was performed according to the manufacturer’s manual. For each experiment, at least 5 fields of view (20× magnification) per dose were counted and only immunoreactivity in nuclei or apoptotic bodies was considered positive.
7
Histopathological Analysis of Mouse Skin Samples
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On the final day of the study (day 14), all mice were sacrificed by cardiac puncture under anaesthesia with isoflurane, and sections of the skin from the anterior neck and chest regions were prepared for histopathology. The skin tissues were placed in cassettes and fixed in 10% formalin. Each sample was further processed using standard methods and embedded in paraffin. 16 The prepared sections (4 mm thick) were stained with haematoxylin (Sigma-Aldrich, St Louis, MO, USA) and eosin (BBC Biochemical, Mt Vernon, WA, USA) (H&E). Tissue slides were evaluated by light microscopy at 100 Â and 400Â magnification. One microphotograph per mouse at 100Â magnification and five microphotographs per mouse at 400Â magnification were obtained. The thickness of the epidermal layer (in micrometres) was calculated from the mean of 10 values per microphotograph at 100Â magnification. The number of inflammatory cells was obtained by counting cells at 400Â magnification. 21 Histopathology on macroscopic and microscopic examinations was blindly performed by an objective veterinary pathologist.
8
Cell Viability in Herbal Extract Media
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Cells were seeded into 6-well culture plates at 1 × 105 cells/ml ratio in seven different media: negative control (I), pin extract (II), ZDBC extract (III), ZDEC extract (IV), HDP extract (V), 5% phenol solution (VI), and reagent control (VII).
Cells were maintained at +37°C ± 1 in a humidified 5% CO2 atmosphere and monitored daily by using an inverted microscope for 72 h in order to evaluate cell morphology and monolayer integrity. After 72 h incubation, haematoxylin/eosin staining was performed. Briefly, media was removed from each well, and cells were washed with PBS and fixed in methanol; 1% haematoxylin (Sigma-Aldrich) solution was added, followed by PBS washing, and 1% eosin staining (Sigma-Aldrich). Cell morphology was evaluated by using an inverted microscope supplied with camera (Leica).
Cells were maintained at +37°C ± 1 in a humidified 5% CO2 atmosphere and monitored daily by using an inverted microscope for 72 h in order to evaluate cell morphology and monolayer integrity. After 72 h incubation, haematoxylin/eosin staining was performed. Briefly, media was removed from each well, and cells were washed with PBS and fixed in methanol; 1% haematoxylin (Sigma-Aldrich) solution was added, followed by PBS washing, and 1% eosin staining (Sigma-Aldrich). Cell morphology was evaluated by using an inverted microscope supplied with camera (Leica).
9
Histological Analysis of Brain and Colon
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Brain and colon sections that had undergone MSI analysis were H and E stained postimaging to permit localisation of candidate metabolites and neurotransmitters to specific brain regions. Sections were fixed on the slide in ice cold 75% acetone and 25% ethanol for 10 min, and air dry for a further 10 min. Slides were placed in water for 2 min, submerged in haematoxylin (Sigma-Aldrich, Poole, Dorset, UK) for 2 min and immediately rinsed in cold running water. The slides were then dipped for 3 sec in acid alcohol 0.5% (Atom Scientific Hyde, Cheshire, UK), and rinsed in water before submerging in Scott's tap water (Atom Scientific Hyde, Cheshire, UK) for a further 30 sec. The sections were counter-stained with eosin (Sigma Aldrich, Poole, Dorset, UK) for 2 min and washed in water. Sections were then dehydrated in increasing concentrations of ethanol (70 % ethanol for 30 sec, 90 % for 1 min, and twice for 3 min in 100 % ethanol), cleared in xylene (twice for 3 min), and cover slipped using DPX mounting media (Atom Scientific, Hyde, Cheshire, UK).
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint this version posted March 14, 2020. ; https://doi.org/10.1101/2020.03.13.987164 doi: bioRxiv preprint
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint this version posted March 14, 2020. ; https://doi.org/10.1101/2020.03.13.987164 doi: bioRxiv preprint
10
Ovarian Follicle Quantification Protocol
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To estimate ovarian follicle numbers, paraffin-embedded ovaries were exhaustively sectioned at 5 μm and stained with periodic acid-Schiff and haematoxylin (n = 5/group) (Sigma-Aldrich). Whole tissue section images were captured on the DotSlide system at ×20 objective using an XC10 camera (Olympus). The total number of primordial, transitional, primary follicles was quantified in every ninth section of each ovary and the total number of secondary and antral follicles were counted in every 36th section using a similar strategy as previously described (Tilly 2003 (link), Hutt et al. 2006) (link). Follicles were counted if the oocyte nucleus was present. Total follicle numbers were obtained by multiplying the raw counts of oocytes sampled (Q-) by nine to correct for the sections not counted. The number of corpora lutea was determined by direct counting of every 36th section encompassing the entire ovary. Adjacent sections were evaluated to ensure each corpora lutea was only counted once.
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