Gill s hematoxylin

Manufactured by Vector Laboratories

Sourced in United States

Gill's hematoxylin is a staining solution used in histology and cytology for the visualization of cell nuclei. It is a commonly used nuclear stain that produces a blue-purple color in nucleic acids.

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Hematoxylin counterstain (Gill’s Formula) (2 citations) Gill’s hematoxylin (H-3401; (2 citations) Hematoxylin Gill’s Formula (8 citations) Hematoxylin (236 citations)

24 protocols using gill s hematoxylin

Quantifying Phosphorylated ERK1/2 in Mouse Brains

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Mouse brains were embedded in paraffin after fixation in 4% paraformaldehyde in PBS overnight at 4 °C. Tissue sections (5 µm) were processed in a citrate buffer for antigen retrieval before immunostaining with phosphorylated ERK1/2 (pERK1/2) Thr202/Tyr204 (CST #9101, 1:200). An Elite ABC kit with NovaRED substrate was used for antibody detection, and tissue sections were counterstained with Gill’s hematoxylin (all from Vector Laboratories, Burlingame, CA, USA). Positively stained cells from a minimum of four images per tumor were quantified using a Nuance spectral unmixing camera and InForm Tissue Finder software (Perkin Elmer, Waltham, MA, USA).

Andradas C., Byrne J., Kuchibhotla M., Ancliffe M., Jones A.C., Carline B., Hii H., Truong A., Storer L.C., Ritzmann T.A., Grundy R.G., Gottardo N.G, & Endersby R. (2021). Assessment of Cannabidiol and Δ9-Tetrahydrocannabiol in Mouse Models of Medulloblastoma and Ependymoma. Cancers, 13(2), 330.

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Immunodetection of 4-HNE Adducts in Liver

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Immunodetection of 4-HNE adducts was conducted as previously described (Roychowdhury et al., 2009b (link)). Liver tissue was formalin-fixed and paraffin-embedded, after which it was sectioned and mounted. Immunohistochemistry was then performed using rabbit antisera against 4-HNE (Alpha diagnostic 1:100). Following primary incubation, sections were conjugated using Vectastain Elite Rabbit IgG Kit (Vector Labs) and visualized using DAB substrate chromogen. Following development, slides were then counterstained using Gill’s hematoxylin (Vector labs). Images were acquired on a 20X objective by a blinded investigator.

Wegner S.A., Pollard K.A., Kharazia V., Darevsky D., Perez L., Roychowdhury S., Xu A., Ron D., Nagy L.E, & Hopf F.W. (2017). Limited excessive voluntary alcohol drinking leads to liver dysfunction in mice. Alcoholism, clinical and experimental research, 41(2), 345-358.

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Quantifying Tau Pathology in Alzheimer's Disease

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Tissue was fixed in periodate-lysine-paraformaldehyde, tissue blocks were paraffin-embedded, and sections were cut at 10μm for immunohistochemistry. Antigen retrieval for and Aβ was performed with formic acid treatment for two minutes. Sections were incubated overnight at 4°C with antibodies to phosphorylated PHF-tau (AT8; Pierce Endogen, Rockford IL; 1:2000) and Aβ (4G8; BioLegend, San Diego, CA; 1:100,000). Sections were washed three times with PBS (pH 7.4), and subsequently treated with biotinylated secondary antibody and labeled with a 3-amino-9-ethylcarbazol HRP substrate kit (Vector Laboratories). The sections were then counterstained with Gill’s Hematoxylin (Vector Laboratories H-3401) and coverslipped using Permount mounting medium. For tau pathology quantification, slides immunostained for ptau (AT8) were scanned at 20× magnification with a Leica Aperio Scanscope (Leica Biosystems, Richmond, IL) as previously described [13 (link)]. ImageScope (Leica Biosystems) was used to highlight the gray matter at the bottom third of the sulcus. Leica’s image analysis and automated counting software (Aperio positive pixel count, Version 9) was calibrated for staining intensity to detect AT8-immunoreactivity within the region of interest. Counts were normalized to the area measured and are presented as positive pixels per mm² within the sulcal depth.

Standring O.J., Friedberg J., Tripodis Y., Chua A.S., Cherry J.D., Alvarez V.E., Huber B.R., Xia W., Mez J., Alosco M.L., Nicks R., Mahar I., Pothast M.J., Gardner H.M., Meng G., Palmisano J.N., Martin B.M., Dwyer B., Kowall N.W., Cantu R.C., Goldstein L.E., Katz D.I., Stern R.A., McKee A.C, & Stein T.D. (2019). Contact sport participation and chronic traumatic encephalopathy are associated with altered severity and distribution of cerebral amyloid angiopathy. Acta neuropathologica, 138(3), 401-413.

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Immunohistochemical Analysis of Tumor Samples

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Fixed tumors and organs were embedded in paraffin and unstained slides were prepared for immunostaining or stained with H&E. IHC staining was performed as previously described (21 (link)). Primary antibodies included rabbit anti-Ki67 (1:400), rabbit anti-CD31 (1:100) (#9027; #77699, Cell Signaling Technology), rabbit anti-cleaved PARP (1:50) and rabbit anti-cleaved-caspase-3 (1:200), detected by a biotinylated horse anti-rabbit IgG antibody (BA-1100, Vector Laboratories Inc., Burlingame, CA) secondary antibody. Anti-human-specific mitochondria IHC staining (AbCAM, Cambridge, MA, cat# ab92824) was performed at a 1:1000 dilution to visualize metastases. All slides were developed with DAB Impact (Vector Labs, Burlingame, CA), counterstained in Gill’s hematoxylin (Vector Labs) and mounted with Cytoseal XYL mounting media. Tissue images were acquired with a Keyence BZ-X700 microscope. Quantification of metastatic burden was performed by digital scanning of whole stained slides using a Pannoramic FLASH III system (3D Histech), followed by manual counting of metastatic lesions present in the tissue section. Quantification of Ki67-, CD31-, cleaved-PARP- and cleaved-caspase-3-positive tumor cells was performed by calculating the area of positive cells in 4–5 representative fields per section using the Keyence Hybrid Cell Count module.

Deng S., Krutilina R.I., Wang Q., Lin Z., Parke D.N., Playa H.C., Chen H., Miller D.D., Seagroves T.N, & Li W. (2019). An orally available tubulin inhibitor, VERU-111, suppresses triple-negative breast cancer tumor growth and metastasis and bypasses taxane resistance. Molecular cancer therapeutics, 19(2), 348-363.

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Immunostaining of NUP62 and pTDP43 in Cortical Tissue

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Staining was performed as previously described (Cherry et al., 2016 (link)). Briefly, tissue blocks of cortical samples were taken from Broadman area 8/9 for all cases. Tissues were embedded in paraffin and cut into 20-µm-thick sections. For chromogenic staining, sections were incubated overnight at 4°C with anti-NUP62 antibody. Sections were treated with biotinylated secondary antibodies and labeled with a 3-amino-9-ethylcarbazol HRP substrate kit (Vector Laboratories). Sections were counterstained with Gill’s hematoxylin (Vector Laboratories, #H-3401). For multiplex immunofluorescent images, sections were incubated overnight at 4°C with anti-NUP62 (BD Transduction Laboratories, #610497) and or anti-pTDP43 (Cosmo Bio, #NC0877946) antibodies. Fluorescent labeling was carried out using the PerkinElmer Opal 7-Color Automation IHC kit (#NEL801001KT), per manufacturer’s instructions. Sections were counterstained with DAPI.

Anderson E.N., Morera A.A., Kour S., Cherry J.D., Ramesh N., Gleixner A., Schwartz J.C., Ebmeier C., Old W., Donnelly C.J., Cheng J.P., Kline A.E., Kofler J., Stein T.D, & Pandey U.B. (2021). Traumatic injury compromises nucleocytoplasmic transport and leads to TDP-43 pathology. eLife, 10, e67587.

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Histological and Immunohistochemical Characterization of TEVG

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Explanted TEVG samples were fixed in 10% formalin for 24 hours at 4°C, and then embedded in paraffin. For the standard histology, staining of tissue sections with hematoxylin and eosin, Masson trichrome, Verhoeff-Van Gieson, and von Kossa stains were done. For the immunohistochemistry, we used the primary antibodies including von Willebrand factor (vWF) (1:2000; Dako, Cat:A0082), α-smooth muscle actin (SMA) (1:500; Abcam, Cat:ab5694), and CD68 (1:200, Abcam,ab31630). Detection of the antibody binding was done by using biotinylated secondary antibodies (Vector Laboratories, Burlingame, Calif), followed by incubation with streptavidinated horseradish peroxidase (Vector Laboratories). The chromogenic reaction with 3,3-diaminobenzidine (Vector Laboratories) was performed for the development of the immunohistochemistry. Counterstaining of the Nuclei was done with Gill’s hematoxylin (Vector Laboratories).

Yeung E., Inoue T., Matsushita H., Opfermann J., Mass P., Aslan S., Johnson J., Nelson K., Kim B., Olivieri L., Krieger A, & Hibino N. (2019). In vivo Implantation of 3D Printed Customized Branched Tissue Engineered Vascular Graft in Porcine Model. The Journal of thoracic and cardiovascular surgery, 159(5), 1971-1981.e1.

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Localization of Hyaluronan in Tumor Tissues

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Hyaluronan in tumor tissues was localized as previously described [42 (link)], with some modifications. Briefly, five micrometer sections were deparaffinized and rehydrated, and endogenous peroxidase was blocked with Peroxo-Block solution (Invitrogen). Nonspecific staining was blocked using 2% BSA (Jackson ImmunoResearch, West Grove, PA) and 2% normal goat serum (Vector) for 1 h, followed by blocking of endogenous avidin and biotin (Avidin/Biotin Blocking Kit, Invitrogen). Hyaluronan was detected by incubating sections with 2.5 μg/mL bHABP (Seikagaku, Tokyo, Japan) for 1 h at 37°C. Signal was amplified by incubation with streptavidin-horseradish peroxidase solution (HRP; BD Biosciences) and detected with 3,3′-diaminobenzidine (DAB, Dako North America, Carpinteria, CA). Sections were then counterstained in Gill's hematoxylin (Vector) and mounted in Cytoseal 60 medium (American MasterTech, Lodi, CA). Specificity of the staining was assessed by incubation of a section of each sample with rHuPH20 (12,000 U/mL) in PIPES buffer (25 mM PIPES, 70 mM NaCl, 0.1% BSA, pH 5.5) at 37°C for 2 h prior to incubation with bHABP.

Kultti A., Zhao C., Singha N.C., Zimmerman S., Osgood R.J., Symons R., Jiang P., Li X., Thompson C.B., Infante J.R., Jacobetz M.A., Tuveson D.A., Frost G.I., Shepard H.M, & Huang Z. (2014). Accumulation of Extracellular Hyaluronan by Hyaluronan Synthase 3 Promotes Tumor Growth and Modulates the Pancreatic Cancer Microenvironment. BioMed Research International, 2014, 817613.

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Histological Analysis of Explanted TEVG

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The middle parts of the explanted TEVG samples were fixed in 10% formalin for 24 hours at 4°C and then embedded in paraffin for standard histologic analysis with hematoxylin and eosin, Masson’s trichrome, Verhoeff-van Gieson, and von Kossa staining. For immunohistochemistry, the tissue sections were deparaffinized, rehydrated, and blocked for endogenous peroxidase activity and nonspecific staining. The primary antibodies used included von Willebrand factor (1:2000; Dako, Agilent Technologies, Santa Clara, Calif), α-smooth muscle actin (α-SMA; 1:500; Dako), and CD68 (1:200; Abcam, Cambridge, UK). Biotinylated secondary antibodies and horseradish peroxidase bound with streptavidin were then used before the color development of the chromogenic reaction with 3,3-diaminobenzidine (Vector Laboratories, Burlingame, Calif). Nuclei counterstaining was performed using Gill’s hematoxylin (Vector Laboratories). The number of cells was measured by magnifying the number of nuclei under hematoxylin and eosin staining and the number of α-SMA–positive cells under immunohistochemical staining 20-fold from the intima to the tunica media tissue. The four samples were then averaged.

Matsushita H., Hayashi H., Nurminsky K., Dunn T., He Y., Pitaktong I., Koda Y., Xu S., Nguyen V., Inoue T., Rodgers D., Nelson K., Johnson J, & Hibino N. (2022). Novel reinforcement of corrugated nanofiber tissue-engineered vascular graft to prevent aneurysm formation for arteriovenous shunts in an ovine model. JVS-Vascular Science, 3, 182-191.

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Immunohistochemical Analysis of Tumor Cryosections

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Solid tumor samples were snap-frozen in OCT medium (Tissue Tek, Sakura, Torrance, CA) and sections were prepared using a Leica CM1950 Cryostat (Leica Microsystems, Bannockburn, IL). Sections were fixed in cold acetone for 10 minutes, pretreated with 3% H₂O₂ for 20 min to block endogenous peroxidase activity and blocked in normal horse serum (Vector Laboratories). Biotinylated goat anti-mouse Exodus-2/CCL21 and goat isotype control (both R&D systems) were used at 1:50 dilution for these studies. Then, the Vectastain ABC kit was applied as described by the manufacturer (Vector Laboratories). Sections were counterstained with Gill's hematoxylin (Vector Laboratories). Images were acquired through a Micropublisher 5.0 Digital CCD Color Camera (Qimaging, Surrey, BC Canada).

Muccioli M., Nandigam H., Loftus T., Singh M., Venkatesh A., Wright J., Pate M., McCall K, & Benencia F. (2018). Modulation of double-stranded RNA pattern recognition receptor signaling in ovarian cancer cells promotes inflammatory queues. Oncotarget, 9(94), 36666-36683.

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Immunohistochemistry and TUNEL Assay for Tissue Analysis

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For immunohistochemistry, paraffin tissue sections were deparaffinized in xylene and hydrated through graded ethanol. Slides were steamed with a Reveal Decloaker (Biocare Medical) to minimize background staining. Sniper Universal Blocking Sera (Biocare Medical) were used throughout the protocol. The slides were stained using anti- Ki67, anti- HSP 70 antibody (Abcam) and anti-GRP78 antibody (Cell signaling). Staining was detected using anti-rabbit secondary antibody conjugated to HRP followed by diaminobenzidine peroxidase (DAB) substrate kit (Vector Laboratories). The tissue sections were counterstained with Gill’s hematoxylin (Vector Laboratories). The primary antibody was omitted for the negative controls. TUNEL was performed using the In situ cell death determination kit (Roche Diagnostics) according to manufacturer’s protocol. For immunofluorescence, fluorescent antibody conjugates were used after primary antibody staining. Slides were counterstained with DAPI and visualized in a Nikon fluorescent microscope. Tissue samples were incubated with mouse IgG1 isotype controls (BD Biosciences) and did not demonstrate any specific staining.

Banerjee S., Nomura A., Sangwan V., Chugh R., Dudeja V., Vickers S.M, & Saluja A. (2014). CD133+ tumor initiating cells (TIC) in a syngenic murine model of pancreatic cancer respond to Minnelide. Clinical cancer research : an official journal of the American Association for Cancer Research, 20(9), 2388-2399.

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