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Hematoxylin counterstaining

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Hematoxylin counterstaining is a laboratory technique used to stain cellular components in histological samples. It is a common staining method employed in various fields, such as pathology and histology, to enhance the contrast and visibility of cellular structures during microscopic examination. The core function of hematoxylin counterstaining is to provide a complementary stain that highlights specific cellular features, enabling better visualization and analysis of the sample.

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Lab products found in correlation

Dab substrate, by Vector Laboratories (3 mentions) Abc elite kit, by Vector Laboratories (3 mentions) Aperio imagescope software, by Leica Biosystems (3 mentions) 3 3 diaminobenzidine (dab), by Vector Laboratories (3 mentions) Aperio scanscope gl, by Leica Biosystems (2 mentions) Phosphatase conjugated streptavidin, by LGC (2 mentions) Histomark red, by LGC (2 mentions) Horseradish peroxidase conjugated secondary anti mouse or anti rabbit antibodies, by Santa Cruz Biotechnology (2 mentions) Cd117 c kit, by BioLegend (1 mentions) Axioplan 2 microscope, by Zeiss (1 mentions) Arid1a, by Merck Group (1 mentions) Anti cleaved caspase 3, by Cell Signaling Technology (1 mentions) Anti ki67 primary antibody, by Cell Signaling Technology (1 mentions) Diaminobenzidine colorimetric reagent solution, by Agilent Technologies (1 mentions) Ab15580, by Cell Signaling Technology (1 mentions) Scanscope gl, by Leica Biosystems (1 mentions) Dab 3 3 diaminobenzidine, by Vector Laboratories (1 mentions) Tris hcl edta ph 9.0 buffer, by Agilent Technologies (1 mentions)

13 protocols using hematoxylin counterstaining

1

Cytological and Histopathological Analysis of Cell Samples

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Cytospin preparations from peripheral blood or bone marrow cells stained with May-Grunwald-Giemsa as described previously (65 (link)). Tissue samples were fixed formalin, dehydrated and embedded in paraffin. Sectioned slides were rehydrated and followed by standard H&E staining protocol. For IHC, antigen was retrieved by boiling slides in 10mM sodium citrate buffer at 90–100°C for 20 minutes and then Cooled down to room temperature. After washing twice with PBST, slides were incubated in methanol with 3% H2O2 for 20 minutes followed by blocking with 5% goat serum. Primary antibody used was c-Kit (CD117) (Biolegend, Cat# 105802). Detection was performed with the Elite ABC Kit and DAB Substrate (Vector Laboratories), followed by hematoxylin counterstaining (Sigma). Reticulin and trichrome staining of BM, spleen or liver sections were performed by the Molecular Pathology Core facility at UTSW.
Gu Z., Liu Y., Cai F., Patrick M., Zmajkovic J., Cao H., Zhang Y., Tasdogan A., Chen M., Qi L., Liu X., Li K., Lyu J., Dickerson K.E., Chen W., Ni M., Merritt M.E., Morrison S.J., Skoda R.C., DeBerardinis R.J, & Xu J. (2019). Loss of EZH2 Reprograms BCAA Metabolism to Drive Leukemic Transformation. Cancer discovery, 9(9), 1228-1247.
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2

Serum Autoantibody Screening in Mice

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Indirect immunohistochemistry was performed on tissue sections from Rag2−/−/γc−/− mice incubated with serum from wt and Was−/− mice (27 (link)). For each serum, we analyzed the reactivity to four tissues (thyroid, stomach, small intestine, and salivary gland and pancreas). Autoantibodies were revealed by using a goat anti-mouse IgG-HRP antibody (Molecular Probes; Invitrogen, Carlsbad, CA, USA) and diaminobenzidine (DBA, DAKO) followed by hematoxylin counterstaining (Sigma-Aldrich). Slides were examined on a Zeiss Axioplan2 microscope (Carl Zeiss Microimaging, Thornwood, NY, USA).
Castiello M.C., Pala F., Sereni L., Draghici E., Inverso D., Sauer A.V., Schena F., Fontana E., Radaelli E., Uva P., Cervantes-Luevano K.E., Benvenuti F., Poliani P.L., Iannacone M., Traggiai E., Villa A, & Bosticardo M. (2017). In Vivo Chronic Stimulation Unveils Autoreactive Potential of Wiskott–Aldrich Syndrome Protein-Deficient B Cells. Frontiers in Immunology, 8, 490.
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3

Immunohistochemical Analysis of Tumor Tissues

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Mice were sacrificed for tumor tissues. Part of tumor tissues were fixed in 10% neutral-buffered formalin, processed and embedded in paraffin. EZH2(1:1000, AR(1:600) and PSA (1:2000) staining were developed using DAB (Vector Laboratories, Burlingame, CA) followed by Hematoxylin counterstaining (Sigma, St. Louis, MO). Detection was developed by Alexa 594 nm conjugated secondary antibodies (Molecular Probes, Eugene, OR) and visualized with microscopes (Daco). The slides were scanned and then quantitated using ImageJ to determine the proportion of stained cells. The results were normalized with the vehicle control group.
Liu Q., Wang G., Li Q., Jiang W., Kim J.S., Wang R., Zhu S., Wang X., Yan L., Yi Y., Zhang L., Meng Q., Li C., Zhao D., Qiao Y., Li Y., Gursel D.B., Chinnaiyan A.M., Chen K, & Cao Q. (2019). Polycomb group proteins EZH2 and EED directly regulate androgen receptor in advanced prostate cancer. International journal of cancer, 145(2), 415-426.
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4

Immunohistochemical Analysis of CBX6, PCNA, and Ki67

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Paraffin-embedded tissue sections and TMAs underwent IHC analyses. Briefly, the slides were probed with primary antibodies specific for the following proteins CBX6, PCNA, and Ki67, and then the slides were treated with anti-rabbit or anti-mouse horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology). Finally, the slides were stained with diaminobenzidine (DAB) colorimetric reagent solution from Dako (Carpinteria, CA, USA) before undergoing hematoxylin counterstaining (Sigma Chemical Co). TMA analysis was performed by scanning the slides with an AperioScanScope GL, and AperioImageScope software (Aperio Technologies, Vista, CA, USA) was used to assess the scanned images by determining the percentages of positively stained cells and staining intensities. CBX6expression in all the clinical samples was quantified, and the tumor CBX1-8 expression level/peri-tumor CBX1-8 expression level ratio was calculated.
Zheng H., Jiang W.H., Tian T., Tan H.S., Chen Y., Qiao G.L., Han J., Huang S.Y., Yang Y., Li S., Wang Z.G., Gao R., Ren H., Xing H., Ni J.S., Wang L.H., Ma L.J, & Zhou W.P. (2017). CBX6 overexpression contributes to tumor progression and is predictive of a poor prognosis in hepatocellular carcinoma. Oncotarget, 8(12), 18872-18884.
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5

Immunohistochemical Analysis of Tissue Samples

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Tissue samples were fixed in 4% paraformaldehyde (PFA) and embedded in paraffin. Primary antibodies used were Ki-67 (Abcam ab15580), p-H3 (Cell Signaling #9706), ARID1A (Sigma HPA005456), V5 (Cell Signaling 13202). Detection was performed with the Elite ABC Kit and DAB Substrate (Vector Laboratories), followed by hematoxylin counterstaining (Sigma).
Sun X., Wang S.C., Wei Y., Luo X., Jia Y., Li L., Gopal P., Zhu M., Nassour I., Chuang J.C., Maples T., Celen C., Nguyen L.H., Wu L., Fu S., Li W., Hui L., Tian F., Ji Y., Zhang S., Sorouri M., Hwang T.H., Letzig L., James L., Wang Z., Yopp A., Singal A.G, & Zhu H. (2017). Arid1a has context-dependent oncogenic and tumor suppressor functions in liver cancer. Cancer cell, 32(5), 574-589.e6.
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6

Quantifying Apoptosis in Tumor Sections

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Paraffin-embedded tumor sections were cut (4 µm), deparaffinized, rehydrated and heated in sodium citrate solution at pH 6.0 for epitope retrieval. 0.5% Triton X-100 was used for permeabilization and 1% bovine serum albumin and 5% goat serum for blocking. Primary antibody staining was carried out with anti-caspase-3 (cleaved) (Cell Signalling, #9661) at 4 °C overnight. This was followed by treatment with biotin-conjugated polyclonal swine anti-rabbit secondary antibody (E0353, Agilent Technologies, Santa Clara, CA, USA) and phosphatase-conjugated streptavidin (Seracare Life Sciences, Milford, MA, USA). For visualization, HistoMark® RED (Seracare Life Sciences) and hematoxylin counterstaining (Sigma-Aldrich) were performed. Quantification was done with ImageJ (Fiji) freeware using the color threshold tool. Caspase-positive cells were counted per area.
Hering N.A., Günzler E., Arndt M., Zibell M., Lauscher J.C., Kreis M.E., Beyer K., Seeliger H, & Pozios I. (2023). Targeting Interleukin-6/Glycoprotein-130 Signaling by Raloxifene or SC144 Enhances Paclitaxel Efficacy in Pancreatic Cancer. Cancers, 15(2), 456.
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7

Quantifying Cellular Proliferation via Ki67

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Tissue sections were prepared and treated as described for immunofluorescence staining. Staining was performed with anti-Ki67 primary antibody (9027, Cell Signaling) at 4 °C overnight followed by biotin-conjugated polyclonal swine anti-rabbit secondary antibody (E0353, Agilent Technologies, Santa Clara, CA, USA) and phosphatase-conjugated streptavidin (Seracare Life Sciences, Milford, CT, USA). Signals were visualized by HistoMark® RED (Seracare Life Sciences) and hematoxylin counterstaining (Sigma-Aldrich). For quantification, Ki67-positive cells were counted per high power field (HPF = 0.159 mm2, 10 HPF counted). The mean of Ki67-positive cells was calculated and expressed in percent.
Hering N.A., Liu V., Kim R., Weixler B., Droeser R.A., Arndt M., Pozios I., Beyer K., Kreis M.E, & Seeliger H. (2021). Blockage of Cholinergic Signaling via Muscarinic Acetylcholine Receptor 3 Inhibits Tumor Growth in Human Colorectal Adenocarcinoma. Cancers, 13(13), 3220.
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8

Immunohistochemical Analysis of Cancer Biomarkers

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Formalin fixed and paraffin-embedded sections (4 μm) were subjected to immunohistochemical staining. The slides were incubated overnight at 4 °C with primary antibodies, including rabbit anti-AHRR (1:100, ab108518, Abcam), rabbit anti-PTPRD(1:100, LS-B9625, LifeSpan Biosciences), rabbit anti-NRG3(1:200, ab83704, Abcam) and mouse anti-UNC5D (1:100, ab58141, Abcam). Anti-rabbit or anti-mouse horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) were applied. Finally, diaminobenzidine colorimetric reagent solution from Dako (Carpinteria, CA) was used and followed by hematoxylin counterstaining (Sigma Chemical Co). Tissue slides were scanned with an Aperio ScanScope GL, and the Aperio ImageScope software (Aperio Technologies, Vista,CA) was used to assess the scanned images based on the percentage of positively stained cells and staining intensity. Expression levels of these proteins in all clinical samples were quantified.
Zhao L.H., Liu X., Yan H.X., Li W.Y., Zeng X., Yang Y., Zhao J., Liu S.P., Zhuang X.H., Lin C., Qin C.J., Zhao Y., Pan Z.Y., Huang G., Liu H., Zhang J., Wang R.Y., Yang Y., Wen W., Lv G.S., Zhang H.L., Wu H., Huang S., Wang M.D., Tang L., Cao H.Z., Wang L., Lee T.L., Jiang H., Tan Y.X., Yuan S.X., Hou G.J., Tao Q.F., Xu Q.G., Zhang X.Q., Wu M.C., Xu X., Wang J., Yang H.M., Zhou W.P, & Wang H.Y. (2016). Genomic and oncogenic preference of HBV integration in hepatocellular carcinoma. Nature Communications, 7, 12992.
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9

Immunohistochemical Analysis of Tissue Samples

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Tissue samples were fixed in 4% paraformaldehyde (PFA) and embedded in paraffin. Primary antibodies against Ki-67 (Abcam, ab15580), PTEN (Cell Signaling, CST#9559S), and FAH (Yecuris, 20–0034) were used. Detection was performed with the Elite ABC Kit and DAB Substrate (Vector Laboratories), followed by hematoxylin counterstaining (Sigma).
Zhu M., Lu T., Jia Y., Luo X., Gopal P., Li L., Odewole M., Renteria V., Singal A.G., Jang Y., Ge K., Wang S.C., Sorouri M., Parekh J.R., MacConmara M.P., Yopp A.C., Wang T, & Zhu H. (2019). Somatic mutations increase hepatic clonal fitness and regeneration in chronic liver disease. Cell, 177(3), 608-621.e12.
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10

Immunohistochemical Analysis of eIF5B, PCNA, and Ki67

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Paraffin-embedded tissue sections and TMAs underwent IHC analyses. Briefly, the slides were probed with primary antibodies specific for the following proteins: eIF5B, PCNA, and Ki67. Anti-rabbit or anti-mouse horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology) were applied. Finally, the slides were stained with diaminobenzidine (DAB) colorimetric reagent solution from Dako (Carpinteria, CA) followed by hematoxylin counterstaining (Sigma Chemical Co). TMA analysis was performed by scanning the slides with an Aperio ScanScope GL, and Aperio ImageScope software (Aperio Technologies, Vista, CA) was used to assess the scanned images based on the percentage of positively stained cells and staining intensity. The expression levels of eIF5B in all clinical samples were quantified, and the ratio of the eIF5B expression level between each tumor/peri-tumor pair was calculated.
Wang Z.G., Zheng H., Gao W., Han J., Cao J.Z., Yang Y., Li S., Gao R., Liu H., Pan Z.Y., Fu S.Y., Gu F.M., Xing H., Ni J.S., Yan H.L., Ren H, & Zhou W.P. (2016). eIF5B increases ASAP1 expression to promote HCC proliferation and invasion. Oncotarget, 7(38), 62327-62339.
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