Same as above. HIER was performed by immersing the tissue sections at 98 °C for 20 min in Envision FLEX High pH retrieval solution from Dako/Agilent (K8004). Briefly, slides were treated as described above, and exposed to primary antibodies for CD4+T cells (1:600, Abcam ab183685) for 1 h at room temperature. Slides were exposed to an anti-rabbit HRP labeled polymer for 30 min and DAB chromagen (Dako/Agilent) for 5 min. Slides were counterstained with Hematoxylin (Fisher, Harris Modified Hematoxylin), blued in 1% ammonium hydroxide, dehydrated, and mounted with Acrymount. Consecutive sections with the primary antibody omitted were used as negative controls.
Harris modified hematoxylin
Manufactured by Thermo Fisher Scientific
Sourced in United States
Harris Modified Hematoxylin is a staining solution used in histology and cytology laboratories. It is a nuclear stain that highlights the nuclei of cells, allowing for the visualization and differentiation of cellular structures. The solution contains hematoxylin, which is a natural dye extracted from the heartwood of the Logwood tree, and other components that enhance the staining process. The stain is commonly used in various tissue preparation and analysis techniques.
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Lab products found in correlation
Dab chromagen, by Agilent Technologies (14 mentions)
Hematoxylin, by Thermo Fisher Scientific (7 mentions)
Permount, by Thermo Fisher Scientific (7 mentions)
Vectastain kit, by Vector Laboratories (6 mentions)
Vectastain abc reagent, by Agilent Technologies (5 mentions)
Eosin y, by Thermo Fisher Scientific (5 mentions)
Avidin biotin blocking kit, by Thermo Fisher Scientific (4 mentions)
Diva decloaker, by Biocare Medical (4 mentions)
K4003, by Agilent Technologies (3 mentions)
Ix71 inverted epifluorescence microscope, by Olympus (3 mentions)
Dab peroxidase substrate kit, by Vector Laboratories (3 mentions)
Vectastain abc, by Vector Laboratories (3 mentions)
Vectastain abc ap kit, by Vector Laboratories (3 mentions)
Vector red alkaline phosphatase substrate kit 1, by Vector Laboratories (3 mentions)
Cryomedium, by Sakura Finetek (3 mentions)
Pt link, by Agilent Technologies (2 mentions)
Dako envision system hrp, by Agilent Technologies (2 mentions)
Hdac2, by Thermo Fisher Scientific (2 mentions)
Vectastain elite abc kit, by Vector Laboratories (2 mentions)
Microtome, by Leica (2 mentions)
62 protocols using harris modified hematoxylin
1
Immunohistochemical Staining of CD4+ T Cells
2
Immunohistochemical Analysis of ASPH
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TMAs of patient tissue samples were stained using anti-ASPH monoclonal antibody (1/100 dilution) and the DAB chromogen kit following the manufacturer’s instructions. Slides were counterstained with Hematoxylin (Fisher, Harris Modified Hematoxylin), blued in 1% ammonium hydroxide, dehydrated, and mounted with Acrymount. Consecutive sections with the primary antibody omitted were used as negative controls. All TMAs were scanned with the Aperio image scope (Aperio GT450) and scored by a pathologist. Staining intensities were classified as 0 = negative, 1 = weak, 2 = moderate, and 3 = strong. The proportion of positive tumor epithelial cells was assessed, and the staining distribution was classified as 0 = no cells, 1 = 1–25% of cells, 2 = 26–50% of cells, and 3 > 50% of cells. Positive fibroblast cells in the stromal compartments were evaluated separately using the same scoring criteria.
3
Immunohistochemical Analysis of Placental OGT
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Five microns sections from formalin fixed paraffin-embedded placenta tissues were de-paraffinized with xylenes and rehydrated through a graded alcohol series. Heat induced epitope retrieval (HIER) was performed by immersing the tissue sections in Target Retrieval Solution, Low pH (DAKO) in the PT Link (DAKO). Immunohistochemical staining was performed using a horseradish peroxidase labeled polymer (Agilent, K4003) according to manufacturer’s instructions. Briefly, slides were either treated with 3% hydrogen peroxide and 10% normal goat serum for 10 min each, and exposed to primary antibody for OGT (1:200, Cell Signaling, D1D8Q) for 1 h at room temperature. Slides were exposed to the appropriate HRP labeled polymer for 30 min and DAB chromagen (Dako) for 5 min. Slides were counterstained with Hematoxylin (Fisher, Harris Modified Hematoxylin), blued in 1% ammonium hydroxide, dehydrated, and mounted with Acrymount. Consecutive sections with the primary antibody omitted were used as negative controls.
4
Immunohistochemical staining for ERRβ in breast cancer
5
Gomori Trichrome Staining for Cryosections
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Cryosectioned tissues were brought to room temperature and immersed in acidified Harris modified hematoxylin (Fisher Scientific) for 5 min. Sections were then washed with running tap water until the water ran clear. Slides were incubated in Gomori trichrome stain (12.8 mM Chromotrope 2R (Sigma), 3.7 mM Fast green FCF (Sigma), 2.1 mM phosphotungstic acid (Sigma), 0.2 M acetic acid (Fisher), deionized water, pH 3.4) for 20 min at room temperature. Excess stain was removed with two quick rinses in freshly made 0.2% acetic acid, and slides immediately placed in 95% ethanol. Tissues were then dehydrated in ascending concentrations of ethanol, cleared and mounted as described above.
6
Lectin Histochemistry for Tissue Analysis
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A detailed description of lectin histochemistry was described previously [35 (link)]. Briefly, the tissue microarray (TMA) slides were deparaffinized and rehydrated through a series of ethanol and water washes. The endogenous peroxidase was blocked using 3% hydrogen peroxide (Sigma Aldrich; St. Louis, MO, USA), followed by an antigen retrieval step (Dako; Santa Clara, CA, USA). Then the tissues were fixed with 4% formaldehyde and permeabilized with 0.5% IGEPAL CA-630 (Fisher Scientific; Pittsburgh, PA, USA). Before lectin staining, the TMA slides were blocked with serum-free protein block (Dako) followed by 0.5 μg/mL biotinylated recombinant AALN224Q lectin with enhanced binding to core-fucosylated structures [2 (link),27 (link)] for 30 min in a humidified chamber. Then, the biotinylated lectin was detected with streptavidin-horseradish peroxidase (Vector Laboratory, Burlingame, CA, USA) and developed with 3.3′-diaminobezidine (DAB) chromogen (Dako). The counterstain was performed with Harris-modified hematoxylin (Fisher Scientific). The image was taken using Hamamatsu Nanozoomer slide scanner, performed by Winship Core Pathology Lab in Emory University, Atlanta, GA, USA, and quantified using the Positive Pixel signal algorithm by Aperio ImageScope, Leica Biosystems Inc, Buffalo Grove, IL, USA.
7
NRIP1 Immunohistochemistry Protocol
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Samples from back skin (3 mm diameter) were excised and fixed overnight in 10% neutral buffered formalin, then transferred to PBS (pH 7.4), and embedded in paraffin. Sections (4 μm thick) of each specimen were cut for H&E staining and IHC study.
Tissue slides were deparaffinized with xylene and then rehydrated with decreasing ethanol concentrations. Antigen retrieval was conducted by boiling-bath method in 0.01 M sodium citrate buffer, pH 6.0 to about 95°C, and then slides were put in the buffer for 15 min. Blocking solution was used to prevent nonspecific binding of antibodies. Sections were incubated with polyclonal anti-NRIP1 antibody (SCBT, Santa Cruz, CA, 1:50 dilution) at 4°C overnight. HRP Detection System (Dako EnVision System HRP; Dako North America, Inc. Carpinteria, CA), was used for detection. After counterstaining with hematoxylin (Harris Modified Hematoxylin, Fisher Scientific, Fairlawn, New Jersey), the sections were dehydrated and mounted. The specific staining of NRIP1 in the sections was examined microscopically (Olympus, Center Valley, PA, USA).
Tissue slides were deparaffinized with xylene and then rehydrated with decreasing ethanol concentrations. Antigen retrieval was conducted by boiling-bath method in 0.01 M sodium citrate buffer, pH 6.0 to about 95°C, and then slides were put in the buffer for 15 min. Blocking solution was used to prevent nonspecific binding of antibodies. Sections were incubated with polyclonal anti-NRIP1 antibody (SCBT, Santa Cruz, CA, 1:50 dilution) at 4°C overnight. HRP Detection System (Dako EnVision System HRP; Dako North America, Inc. Carpinteria, CA), was used for detection. After counterstaining with hematoxylin (Harris Modified Hematoxylin, Fisher Scientific, Fairlawn, New Jersey), the sections were dehydrated and mounted. The specific staining of NRIP1 in the sections was examined microscopically (Olympus, Center Valley, PA, USA).
8
Tissue Fixation and Histochemical Staining
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HIO and tHIO tissues were fixed in 4% paraformaldehyde (Sigma) overnight and then dehydrated in an alcohol series: 30 min each in 25%, 50%, 75% Methanol:PBS/0.05% Tween 20, followed by 100% methanol, and then 100% ethanol. Tissue was processed into paraffin using an automated tissue processor (Leica ASP300). Paraffin blocks were sectioned 7-μM thick, and immunohistochemical staining was performed as previously described (Spence et al., 2009 (link)). A list of antibody information and concentrations used can be found in the Supplemental Experimental Procedures . H&E staining was performed using Harris Modified Hematoxylin (FisherScientific) and Shandon Eosin Y (ThermoScientific) according to the manufacturer's instructions. Alcian blue/PAS staining was performed using the Newcomer Supply Alcian blue/PAS Stain kit (Newcomer Supply, Inc.) according to manufacturer's instructions. Trichrome staining was performed by the University of Michigan in vivo Animal Core.
9
Immunohistochemical Staining of STAG2
10
Immunohistochemical Analysis of TRPV Channels in Myometrium
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Paraffin-embedded myometrial tissue sections were freed from paraffin and rehydrated before immunohistochemical staining was performed according to the standard avidin-biotin immunoperoxidase complex technique using the following antibodies: Anti-TRPV1 (Alomone Labs, Jerusalem, Israel; cat#ACC-030; dilution 1:200), anti-TRPV3 (Abcam, Cambridge, UK; cat #ab231150; dilution 1:200), anti-TRPV4 (Abcam; cat #ab39260 dilution 1:200) or rabbit isotype IgG (Dako; Glostrup Denmark; Cat#X0903) as negative control. Diaminobenzidine (DAB; Sigma Fast kit, Sigma-Aldrich; Oakville ON Canada; cat#D4168) was used for the detection of the labeled proteins, and the sections were counterstained with Harris modified hematoxylin (Fisher Scientific, Cat#SH26−500D). Slides were scanned with a Hamamatsu Nanozoomer 2.0-RS scanner from the Histology and Electron Microscopy platform at the Faculty of Medicine and Health Sciences of the Université de Sherbrooke.
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