Xylene substitute mountant

Manufactured by Thermo Fisher Scientific

Sourced in Germany

Xylene Substitute Mountant is a mounting medium used in histological and microscopy applications. It is designed to replace xylene, a commonly used solvent, while providing similar optical properties for mounting and preserving specimens.

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Lab products found in correlation

8 protocols using xylene substitute mountant

In Vitro Endothelial and Smooth Muscle Cell Assays

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Thrombin, benzonase nuclease (DNase), and bicinchoninic acid (BCA) protein assay kit were obtained from Sigma-Aldrich. Calcein AM, Live/Dead Cell Viability Assay, glycine, bovine serum albumin (BSA), fetal bovine serum (FBS), trypsin–EDTA, Medium 200 (M200), Medium 231 (M231), Low Serum Growth Supplement kit (LSGS), Xylene Substitute Mountant, ProLong Diamond Anti-fade Mountant with DAPI, goat anti-rabbit IgG-Alexa Fluor 546 antibody, and α-smooth muscle actin antibody-Alexa Fluor 488 antibody (α-SMA) were obtained from Thermo-Fisher Scientific. Nucleic acid sequences (Table S1) were obtained from Integrated DNA Technologies (Coralville, IA). 4% paraformaldehyde solution and human Fg were obtained from Millipore (Billerica, MA). Vascular endothelial growth factor-165 (VEGF), platelet-derived growth factor-BB (PDGF-BB), VEGF enzyme-linked immunosorbent assay (ELISA) kit, and PDGF-BB ELISA kit were obtained from PeproTech (Rocky Hill, NJ). Primary rabbit anti-mouse CD31 was obtained from Cell Signaling Technology (Beverly, MA). H&E stain kit was obtained from LeicaBiosystems (Buffalo Grove, IL). Human umbilical vein endothelial cells (HUVECs) were obtained from Thermo-Fisher Scientific. Human aorta smooth muscle cells (HASMCs) were obtained from ATCC (Manassas, VA).

Zhao N., Suzuki A., Zhang X., Shi P., Abune L., Coyne J., Jia H., Xiong N., Zhang G, & Wang Y. (2019). Dual Aptamer-functionalized In Situ Injectable Fibrin Hydrogel for Promotion of Angiogenesis via Co-delivery of VEGF and PDGF-BB. ACS applied materials & interfaces, 11(20), 18123-18132.

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Immunohistochemical Analysis of γH2AFX Foci

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Immunohistochemistry was performed on 4 μ-sections of paraffin-fixed embedded tissue. Antigen retrieval was performed with citrate buffer (pH 6) using a Microwave Tender Cooker (Nordic Ware) and a 700 W microwave (15 min 700 W/15 min 350 W). Endogenous peroxidase activity was inhibited with 0.3% H₂O₂ in methanol (25 min) and after that the sections were permeabilized and blocked simultaneously with PBS + 1%BSA + 0,1% TX-100, during 30 min. The slides were then washed once with PBS for 5 min. Endogenous biotin, biotin receptors and avidin binding sites were blocked by using the avidin/biotin blocking kit from Vector Laboratories (cat.#SP-2001). The slides were washed with PBS and incubated ON, in a humidity chamber, with the primary antibody diluted in blocking solution (PBS + 1%BSA). Incubations with the secondary biotinylated antibodies and development were performed with the kits MP-7402 (ImmPRESS) and SK-4605 (ImmPACT VIP HRP substrate) from Vector Laboratories, following their instructions. Methyl green (cat.#M8884-5G; Sigma-Aldrich) was used as counterstaining and xylene substitute mountant (cat.#1,900,231; Thermofisher) as mounting medium. Primary antibody used was γH2AFX (1:1000, cat.#05–636 Millipore). Damaged cells were counted from representative immunohistochemical fields for each experimental condition.

Guijarro T., Magro-Lopez E., Manso J., Garcia-Martinez R., Fernandez-Aceñero M.J., Liste I, & Zambrano A. (2018). Detrimental pro-senescence effects of vitamin D on lung fibrosis. Molecular Medicine, 24, 64.

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Histochemical Analysis of Tissue Spheroids

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All groups of TSs were cultured for 3 weeks, de-crosslinked, fixed with 4% paraformaldehyde, and embedded in paraffin using an automatic tissue processor (TP 1020, Leica, Germany). Next, TSs were gradually dehydrated in alcohol, cut into 8 μm sections, and placed onto charged slides. Sections were then dewaxed using Leica Autostainer XL (Leica, Germany) and underwent Toluidine Blue O and Picrosirius Red/Fast Green staining according to standard protocols for detection of GAGs and collagen, separately. Briefly, for Toluidine Blue O staining, sections were incubated in a Toluidine Blue solution (0.1% in DI water, Sigma Aldrich, MO) at room temperature for 2 min.^{[52 ]} The dye was then removed and samples were washed twice with DI water. After dehydration with ascending alcohol and clearing with xylene, coverslips were mounted to the slides using Xylene Substitute Mountant (Thermo Fisher Scientific Inc., PA). Picrosirius Red/Fast Green staining solution was prepared by dissolving 0.1% direct Red 80 and 0.1% Fast Green FCF in saturated aqueous picric acid (1.2% picric acid in water, Sigma Aldrich, MO). Picrosirius Red/Fast Green solution was then applied to the sections and sections were incubated for 1 h.^{[53 (link)]} Samples were rinsed with DI water, dehydrated with alcohol, cleaned with xylene, and then mounted. Finally, samples were imaged using a BX51 microscope (Olympus, Japan).

Wu Y., Ayan B., Moncal K.K., Kang Y., Dhawan A., Koduru S., Ravnic D.J., Kamal F, & Ozbolat I.T. (2020). Hybrid Bioprinting of Zonally-stratified Human Articular Cartilage Using Scaffold-free Tissue Strands as Building Blocks. Advanced healthcare materials, 9(22), e2001657.

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Immunohistochemical Analysis of EREG

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Paraffin‐embedded tissues were deparaffinized with xylene and rehydrated in a descending alcohol series (100%, 96% and 70%). Slices were boiled in citrate buffer (pH = 6), blocked with 1% BSA in PBS (60 min, room temperature) and incubated overnight at 4°C in 0.5% BSA in PBS containing 10 μg/mL α-EREG (goat, polyclonal, R&D Systems, Minneapolis, Minnesota, US) antibody. After three washing steps with PBS, slices were incubated (1 h, room temperature) in 0.5% BSA in PBS with 1:200 diluted biotinylated α-goat (rabbit, polyclonal, Dako, Agilent Technologies, Waldbronn, Germany) antibody. After washing with PBS three times, signal enhancement was achieved by Vectastain^® (Linaris, Wertheim‐Bettingen, Germany) according to the manufacturer’s instructions. Immunoreaction was visualized with diaminobenzidine (DAB) (Sigma-Aldrich, Hamburg, Germany), followed by nuclear staining using hematoxylin solution (Thermo Fisher Scientific, Oberhausen, Germany). Finally, slices were dehydrated and mounted with Xylene Substitute Mountant (Thermo Fisher Scientific, Oberhausen, Germany). Slides were scanned using a Aperio ScanScope Slide Scanner (Leica Biosystems, Nußloch, Germany) and saved as ScanScope Virtual Slide (.svs) files.

Wiesehöfer M., Raczinski B.B., Wiesehöfer C., Dankert J.T., Czyrnik E.D., Spahn M., Kruithof-de Julio M, & Wennemuth G. (2023). Epiregulin expression and secretion is increased in castration-resistant prostate cancer. Frontiers in Oncology, 13, 1107021.

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Immunohistochemical Analysis of Chromogranin A and Synaptophysin

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Paraffin-embedded tissue blocks were cut at a thickness of 7 µm and slices were mounted on glass slides, deparaffinized with xylene and rehydrated in a descending alcohol series (100%, 96% and 70%). Slices were boiled in citrate buffer for antigen unmasking, were blocked for 60 min at room temperature with 1% BSA in PBS and were incubated overnight at 4 °C with rabbit anti-CHGA or anti-SYP antibody (1:200 in PBS with 0.5% BSA). After three washing steps with PBST, slices were incubated for 1 h at room temperature in PBS/0.5% BSA with diluted (1:200) secondary biotinylated swine anti-rabbit IgG. After washing three times with PBST, signal enhancement was achieved by Vectastain^® (Linaris, Wertheim-Bettingen, Germany) according to the manufacturer instructions. Immunoreaction was visualized with diaminobenzidine (DAB) (Sigma), followed by nuclear staining using hematoxylin solution (Thermo Fisher Scientific, Oberhausen, Germany). Finally, slices were dehydrated and mounted with Xylene Substitute Mountant (Thermo Fisher Scientific, Oberhausen, Germany). Slides were scanned using a Leica DM4000 B microscope, Leica DFC290 camera and analyzed using the Leica Application Suite version 2.8.1 (Leica Microsystems, Wetzlar, Germany).

Wiesehöfer M., Czyrnik E.D., Spahn M., Ting S., Reis H., Dankert J.T, & Wennemuth G. (2021). Increased Expression of AKT3 in Neuroendocrine Differentiated Prostate Cancer Cells Alters the Response Towards Anti-Androgen Treatment. Cancers, 13(3), 578.

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Histological Characterization of Doublet Structures

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To confirm morphologies of doublet structures, sectioned samples were also stained with H&E. Doublet structures for all groups for both strategies were fixed after 7, 14, and 21 d of culture in 10% neutral buffered formalin overnight and gradually dehydrated in alcohol. Samples were then embedded in paraffin blocks using the Leica TP 1020 automatic tissue processor and cut into 10 μm sections with the Shandon Finesse® Paraffin microtome. After collecting the sections on glass microscope slides, samples were placed in a Leica Autostainer XL (Leica, Germany) based on the manufacturer’s protocol. After the staining process, stained sections were mounted using a xylene substitute mountant (Thermo Scientific, Waltham, MA) and kept at room temperature to dry overnight. Stained samples were imaged using an EVOS microscope (Invitrogen, MA).

Celik N., Kim M.H., Hayes D.J, & Ozbolat I.T. (2021). miRNA induced co-differentiation and cross-talk of adipose tissue-derived progenitor cells for 3D heterotypic pre-vascularized bone formation. Biofabrication, 13(4), 10.1088/1758-5090/ac23ae.

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Masson's Trichrome Staining of Cardiac Tissue

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For Masson Trichrome (MT) staining, paraffin embedded ventricular sections (5 µm) were dehydrated through a series of histosolve (Thermo Scientific) and graded alcohols and incubated in biebrich scarlet-acid fuchsin solution to stain cellular material, and subsequently incubated in a phosphomolybdic–phosphotungstic acid solution, aniline blue, and finally 1% acetic acid to stain the collagen fibers. After staining, sections were mounted with a xylene substitute mountant (Thermo Scientific) and covered with a cover slip. Photomicrographs (×400) were taken with the use of a Leica DMLB light microscope (Leica Microsystems, Milton Keynes, Bucks, UK) and collagen volume fraction (CVF) was calculated by determining the percentage area of blue (collagen) stained tissue within 10 fields randomly selected from the left ventricle using computerised planimetry (ImageJ software, National Institute of Health (NIH), Rockville Pike Bethesda, MD). Briefly, images were converted to RGB stacks, separated into a montage, and the red channel thresholded to detect the stained collagen (http://rsbweb.nih.gov/ij/docs/examples/stained-sections/index.html). For continuity, the same threshold was used on images from all animals and the measurement of collagen restricted to the left ventricle to correspond with the functional pressure volume loop data.

Walsh S.K., Hector E.E., Andréasson A.C., Jönsson-Rylander A.C, & Wainwright C.L. (2014). GPR55 Deletion in Mice Leads to Age-Related Ventricular Dysfunction and Impaired Adrenoceptor-Mediated Inotropic Responses. PLoS ONE, 9(10), e108999.

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Histological Examination of Spheroids

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Sectioned samples of single spheroids and bioprinted tissues were stained with H&E using a Leica Autostainer ST5010 XL (Leica, Germany) based on the manufacturer’s protocol. After the staining process, sections were mounted using a Xylene Substitute Mountant (Thermo Fisher Scientific, Waltham, MA) and kept at room temperature to dry overnight. Stained samples were imaged using an EVOS fluorescence microscope (Invitrogen, Waltham, MA).

Celik N., Kim M.H., Yeo M., Kamal F., Hayes D.J, & Ozbolat I.T. (2022). miRNA Induced 3D Bioprinted-Heterotypic Osteochondral Interface. Biofabrication, 14(4), 10.1088/1758-5090/ac7fbb.

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