Histoclear

Manufactured by Thermo Fisher Scientific

Sourced in United Kingdom, United States

Histoclear is a laboratory solvent used in the preparation of tissue samples for histological analysis. It is a clear, colorless liquid that serves as a clearing agent, facilitating the removal of paraffin or other embedding media from tissue sections prior to staining and microscopic examination.

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Lab products found in correlation

Histoclear, by National Diagnostics (4 mentions) Permount, by Thermo Fisher Scientific (3 mentions) Picrosirius red, by Merck Group (2 mentions) Donkey serum, by Jackson ImmunoResearch (2 mentions) Histogel, by Thermo Fisher Scientific (2 mentions) Permount mounting medium, by Thermo Fisher Scientific (2 mentions) Plastic moulds, by CellPath (2 mentions) Charged superfrost microscope slides, by Thermo Fisher Scientific (2 mentions) Harris hematoxylin solution counterstain, by Merck Group (2 mentions) Goat anti mouse cd8 antibody, by Abcam (2 mentions) Histofine simple stain aec solution, by Nichirei Biosciences (2 mentions) Mayer s haematoxylin, by Merck Group (2 mentions) Paraformaldehyde (pfa), by Electron Microscopy Sciences (2 mentions) Microtome, by Leica (2 mentions) 4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions) Triton x 100, by Merck Group (2 mentions) Em 9400, by Jackson ImmunoResearch (1 mentions) H 1200, by Vector Laboratories (1 mentions) Ix51 microscope, by Olympus (1 mentions) Hematoxylin qs, by Vector Laboratories (1 mentions)

Spelling variants of this product

HistoClear solution (2 citations)

42 protocols using histoclear

Preparing Epidermal Equivalents for H&E Staining

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Epidermal equivalents were fixed in 10% formalin (Sigma-Aldrich, Gillingham, UK) in PBS overnight at 4 °C then gradually dehydrated in 30–100% ethanol and Histoclear (ThermoFisher Scientific, Loughborough, UK) before being embedded in paraffin wax using dispomoulds (CellPath, Newton, UK). All wax blocks were sectioned at 7 μm using a Leica RM2125RT microtome and mounted onto Superfrost plus microscope slides (4951PLUS4, ThermoFisher Scientific, Loughborough, UK).
For haematoxylin and eosin (H and E) staining, slides were deparaffinised in Histoclear then gradually rehydrated in ethanol (100–70%) and deionised water. The slides were incubated in Mayer’s haematoxylin (Sigma-Aldrich, Gillingham, UK) for 5 min, then submerged in deionised water followed by alkaline ethanol for 30 s. Samples were once again dehydrated through submersion in ethanol before being incubated in eosin (Sigma-Aldrich, Gillingham, UK) for 20 s and further dehydrated in ethanol and Histoclear. Slides were mounted using DPX (Sigma-Aldrich, Gillingham, UK) and imaged using a Leica ICC50 high-definition camera mounted onto a Brightfield Leica microscope.

Hunter-Featherstone E., Young N., Chamberlain K., Cubillas P., Hulette B., Wei X., Tiesman J.P., Bascom C.C., Benham A.M., Goldberg M.W., Saretzki G, & Karakesisoglou I. (2021). Culturing Keratinocytes on Biomimetic Substrates Facilitates Improved Epidermal Assembly In Vitro. Cells, 10(5), 1177.

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Histological Staining of Adipose and Liver Tissues

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Adipose or liver tissues were fixed in 10% neutral buffered formalin (#245684, FisherScientific) for 24 h and embedded in paraffin or subsequently infiltrated with 30% sucrose for a further 48 h before being frozen in Tissue-Tek optimal cutting temperature solution (#4583, Sakura). Paraffin sections were stained for H&E, or αSMA by Virgnia Commonwealth University Tissue and Data Acquisition and Analysis Core. For staining of collagen fibres, paraffin sections were dewaxed and rehydrated by submerging in Histoclear (#5089990147, FisherScientific), Histoclear 1:1 with ethanol then ethanol for 10 min, 50% ethanol/distilled water then distilled water each for 5 min. Sections were then stained with Sirius red/Fast green (#9046, Chondrex). Frozen liver sections were also stained with oil red O (#O0625, Sigma) to assess neutral lipid accumulation as previously described [22 (link)]. Color deconvolution by ImageJ was used to measure the positive areas of Sirius red, αSMA, and oil red O stained images.

Brown R.D., Green C.D., Weigel C., Ni B., Celi F.S., Proia R.L, & Spiegel S. (2023). Overexpression of ORMDL3 confers sexual dimorphism in diet-induced non-alcoholic steatohepatitis. Molecular Metabolism, 79, 101851.

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Immunohistochemical Analysis of Kidney Samples

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Cell clusters or excised kidneys were fixed with 4% paraformaldehyde (PFA; Electron Microscopy Science; 15,714) for 1 h at room temperature (RT) or overnight at 4 °C, respectively, before being paraffin embedded and sectioned at 10 μm. Paraffin was removed with Histoclear (Thermo Scientific; C78-2-G) and rehydrated, and antigens retrieved by 2 h 0.1 M EDTA (Ambion; AM9261) treatment in a pressure cooker (Proteogenix; 2,100 Retreiver). Samples were blocked with 0.1% Triton X-100 (VWR; EM-9400) and 5% donkey serum (Jackson Immunoresearch; 017-000-121) in PBS (staining solution) for 1 h at RT, incubated with primary antibodies diluted in staining solution overnight at 4 °C, washed for 5 min in staining solution, incubated covered with appropriate Alexa Fluor-488 or -594 secondary antibodies diluted 1:300 in staining solution 2 h at RT, washed for 5 min in staining solution, mounted with Vectashield (Vector Laboratories; H-1200) and covered with a coverslip. Images were taken with an Olympus IX51 Microscope. The primary antibodies used to assess differentiation throughout this study are given in Supplementary Table 6 (ref. 17 (link)).

Millman J.R., Xie C., Van Dervort A., Gürtler M., Pagliuca F.W, & Melton D.A. (2016). Generation of stem cell-derived β-cells from patients with type 1 diabetes. Nature Communications, 7, 11463.

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Comprehensive Tissue Histological Analysis

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Paraffin-embedded tissue sections were stained with haematoxylin and eosin (H&E; Leica, Germany), Picrosirius Red (Merck Sigma Aldrich., Germany) for collagen, Elastin Van Gieson (Millers Elastin, TCS Biosciences, UK) for elastin, and Alcian Blue (AB; BDH Chemicals Ltd, Cellpath Ltd) stains for glycosaminoglycans (GAG). Appropriate positive controls were used to ensure histological stains were performed correctly (small intestine for PR and AB, lung for EVG). For all histological samples, three biological and technical replicates were assessed using standard H&E prior to any special stains to ensure homogeneity. All images shown are representative samples.
For H&E staining, cryosections of 3D cultures and 2D cultured cells on matrigel-coated dishes were fixed in 4% PFA in PBS 15 min, rinse with PBS followed by tap water. They were incubated with Hematoxylin QS (Vector Laboratories, UK) 1–2 min, rinsed with tap water 5 min, and incubated with Eosin Y solution (Sigma-Aldrich, UK) for 30 sec. They were dehydrated with 95% then 100% EtOH, dipped in Histoclear (ThermoFisher scientific, UK) and mounted in a non-aqueous mounting medium.

Lorvellec M., Scottoni F., Crowley C., Fiadeiro R., Maghsoudlou P., Pellegata A.F., Mazzacuva F., Gjinovci A., Lyne A.M., Zulini J., Little D., Mosaku O., Kelly D., De Coppi P, & Gissen P. (2017). Mouse decellularised liver scaffold improves human embryonic and induced pluripotent stem cells differentiation into hepatocyte-like cells. PLoS ONE, 12(12), e0189586.

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Histological Sectioning of Whole Mount ISH Larvae

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Sections were prepared from the whole mount
ISH larvae. The larvae were dehydrated in graded
ethanol, and embedded in wax using Histoclear
(Thermo Scientific, USA) as the intermediate
reagent. Some sections were prepared
with Technovit (Technovit, Kulzer Heraus, Germany)
embedding after dehydration. The thickness
of sections was 5 μm.

Sharif F., de Bakker M.A, & Richardson M.K. (2014). Osteoclast-like Cells in Early Zebrafish Embryos. Cell Journal (Yakhteh), 16(2), 211-224.

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Immunostaining of Pancreatic Islets

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Clusters were fixed with 4% paraformaldehyde (Electron Microscopy Science; 15714) overnight at 4 °C, embedded in Histogel (Thermo Scientific; hg-4000–012), and paraffin-embedded and sectioned by the Division of Comparative Medicine (DCM) Research Animal Diagnostic Laboratory Core at Washington University. Immunostaining was performed by paraffin removal with Histoclear (Thermo Scientific; C78–2-G), rehydration by treatment with increasing ratios of water to ethanol, antigens retrieved by treatmetn with 0.05 M EDTA (Ambion; AM9261) in a pressure cooker (Proteogenix; 2100 Retriever). Non-specific antibody binding was blocked with a 30-min treatment in staining buffer (5% donkey serum (Jackson Immunoresearch; 017–000-121) and 0.1% Triton-X 100 (Acros Organics; 327371000) in PBS), followed by overnight staining with 1:300 dilutions of rat-anti-C-peptide (DSHB; GN-ID4-S) and mouse-anti-glucagon (ABCAM; ab82270) primary antibodies or only with buffer. Samples were stained with donkey secondary antibodies containing Alexa Fluor fluorophores (Invitrogen) for 2 hr at 4 °C, and treated with DAPI in the mounting solution Fluoromount-G (SouthernBiotech; 0100–20). Imaging was performed on a Nikon A1Rsi confocal microscope.

Augsornworawat P., Velazco-Cruz L., Song J, & Millman J.R. (2019). Hydrogel platform for in vitro three dimensional assembly of human stem cell-derived islet cells and endothelial cells. Acta biomaterialia, 97, 272-280.

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Paraffin Embedding of PFA-Fixed Brains

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PFA-fixed brains were paraffin-embedded using a Shandon Excelsior (Thermo Fisher Scientific, Massachusetts, United States of America). Brains were placed in the following solutions in a stepwise fashion, under vacuum conditions: 70% ethanol (90 minutes), 80% ethanol (90 minutes), 90% ethanol (90 minutes), 100% ethanol (3 steps of 90 minutes each), Histoclear (Thermo Fisher Scientific, Massachusetts, United States of America; 2 steps of 90 minutes each, and 1 step of 120 minutes), and finally paraffin wax (2 steps of 60 minutes and 1 step of 120 minutes). Paraffin blocks were sectioned at 4 μM using a Leica microtome (Leica Microsystems, Wetzlar, Germany), and sections mounted on slides.

Hunn B.H., Vingill S., Threlfell S., Alegre-Abarrategui J., Magdelyns M., Deltheil T., Bengoa-Vergniory N., Oliver P.L., Cioroch M., Doig N.M., Bannerman D.M., Cragg S.J, & Wade-Martins R. (2019). Impairment of Macroautophagy in Dopamine Neurons Has Opposing Effects on Parkinsonian Pathology and Behavior. Cell Reports, 29(4), 920-931.e7.

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Quantifying Myocardial Fibrosis with PicroSirius Red

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To measure the extent of myocardial fibrosis, paraffin‐embedded sections of the heart were stained using PicroSirius Red (Cat# 365548, Sigma‐Aldrich, St. Louis, MO). The paraffin‐embedded sections were then deparaffinized using Histo‐Clear (National Diagnostics, Atlanta, GA) and rehydrated using an alcohol gradient (2× 100%, followed by 90%, 80%, and 70%). The sections were washed in distilled water and stained with PicroSirius Red for 1 hour before being washed twice in acidified water for 3 minutes each. The slides were then placed twice in 100% alcohol and Histo‐Clear and mounted using Permount mounting medium (Thermo Fisher Scientific, Waltham, MA). Tissue images were taken at ×400 magnification using an Olympus BH2 microscope. ImageJ software (NIH, Bethesda, MD) was used to measure the degree of fibrosis.

Thirunavukkarasu M., Selvaraju V., Joshi M., Coca‐Soliz V., Tapias L., Saad I., Fournier C., Husain A., Campbell J., Yee S., Sanchez J.A., Palesty J.A., McFadden D.W, & Maulik N. (2018). Disruption of VEGF Mediated Flk‐1 Signaling Leads to a Gradual Loss of Vessel Health and Cardiac Function During Myocardial Infarction: Potential Therapy With Pellino‐1. Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease, 7(18), e007601.

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In Situ Analysis of Hedgehog Signaling

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Mouse EHBD formalin‐fixed paraffin‐embedded sections (5 μm) were deparaffinized and rehydrated in Histoclear (National Diagnostics, Atlanta, GA) and 100% ethanol, followed by 10 minutes of incubation in H₂O₂ (RNAscope Pretreatment Reagents, catalog #322330; ACD, Newark, CA). Next, slides were incubated in RNAScope Target Retrieval Reagent (catalog #322000; ACD) for 15 minutes at 95°C, rinsed with water and 100% ethanol, and treated with RNAScope Protease Plus (ACD) for 30 minutes at 40°C. Slides were then incubated with RNAScope probes (negative control [catalog #310043; ACD], Mm‐Shh [catalog #314361; ACD], Mm‐Ihh [catalog #413091; ACD], or Mm‐Gli1 [catalog #311001; ACD]) for 2 hours, with signal amplification using RNAscope 2.5 HD Detection Reagent (ACD) for 60 minutes at 40°C with washes using RNAscope Wash Buffer (ACD). Slides were incubated with 3,3′‐diaminobenzidine for 10 minutes, counterstained with 1:8 Harris’ hematoxylin (Thermo Fisher Scientific), dehydrated in graded ethanol and Histoclear, and mounted with Permount.

Razumilava N., Shiota J., Mohamad Zaki N.H., Ocadiz‐Ruiz R., Cieslak C.M., Zakharia K., Allen B.L., Gores G.J., Samuelson L.C, & Merchant J.L. (2018). Hedgehog Signaling Modulates Interleukin‐33‐Dependent Extrahepatic Bile Duct Cell Proliferation in Mice. Hepatology Communications, 3(2), 277-292.

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Histological Analysis of Differentiated Cells

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Differentiated cell clusters or islets were fixed with 4% PFA for 1 hr at room temperature (RT), washed, embedded in Histogel (Thermo), and sectioned for histological analysis. Before staining, paraffin was removed from sections using Histoclear (Thermoscientific), rehydrated, and underwent antigen retrieval by treatment with 0.1M EDTA (Ambion) in pressure cooker (Proteogenix) for 2 hr. For staining, slides were blocked with PBS+0.1% Triton X-100 (VWR)+5% donkey serum (Jackson Immunoresearch) for 1 hr at RT, incubated with primary antibodies overnight at 4°C, washed, incubated with secondary antibody incubation for 2 hr at RT, and washed. For imaging, samples were mounted in Vectashield (Vector Laboratories), covered with coverslips, and sealed with nail polish. Representative images were taken using an Olympus IX51 Microscope or Zeiss LSM 510 or 710 confocal microscope.

Pagliuca F.W., Millman J.R., Gürtler M., Segel M., Van Dervort A., Ryu J.H., Peterson Q.P., Greiner D, & Melton D.A. (2014). Generation of functional human pancreatic β cells in vitro. Cell, 159(2), 428-439.

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