A total of 3-μm-thick consecutive tissue sections were prepared from formalin-fixed and paraffin-embedded tissues. Deparaffinization, rehydration, antigen retrieval, and peroxidase blocking were performed on a fully automated system BENCHMARK ULTRA (Roche) according to manufacturer recommendations. The sections were incubated with the following primary antibodies: anti-CD68 clone Kp1 (Dako) and anti-DPD (Thermo Fisher Scientific). Revelation was performed using the Ultraview DAB revelation kit (Roche). Nuclei were counterstained with hematoxylin solution (Dako). Images were captured using an APERIO ATS scanner (Leica).
Hematoxylin solution
Manufactured by Agilent Technologies
Sourced in Denmark
Hematoxylin solution is a laboratory reagent used in histology and cytology for staining biological samples. It is a purplish-blue dye that selectively stains cell nuclei, allowing for the visualization and identification of cellular structures under a microscope.
Automatically generated - may contain errors
Lab products found in correlation
Diaminobenzidine tetrahydrochloride, by Biocare Medical (2 mentions)
Edta buffer, by Agilent Technologies (2 mentions)
Anti cd68 clone kp 1, by Agilent Technologies (1 mentions)
Ultraview dab revelation kit, by Roche (1 mentions)
Benchmark ultra, by Roche (1 mentions)
Citrate buffer, by Merck Group (1 mentions)
Kp 1 clone, by Agilent Technologies (1 mentions)
Clone l26, by Agilent Technologies (1 mentions)
Mach 1, by Biocare Medical (1 mentions)
Af 317 na, by R&D Systems (1 mentions)
Permafluor, by Thermo Fisher Scientific (1 mentions)
Oil red o, by Merck Group (1 mentions)
Optical microscope, by Olympus (1 mentions)
Biotinylated secondary antibody, by Vector Laboratories (1 mentions)
Abc reagent, by Vector Laboratories (1 mentions)
3 3 diaminobenzidine tetrahydrochloride hydrate dab, by Merck Group (1 mentions)
Background sniper, by Biocare Medical (1 mentions)
Ferangi blue, by Biocare Medical (1 mentions)
3 3 diaminobenzidine (dab), by Biocare Medical (1 mentions)
Anti human cd68, by Agilent Technologies (1 mentions)
8 protocols using hematoxylin solution
1
Immunohistochemical Analysis of CD68 and DPD
2
Immunohistochemistry Protocol for Tissue Sections
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2 μm-thick consecutive tissue sections were prepared from formalin-fixed and paraffin-embedded tissues, provided by the Pathology Department of the Humanitas Clinical and Research Center, and processed for immunohistochemistry. Briefly, after deparaffinization and rehydration, antigen retrieval was performed by heat treatment using EDTA buffer (0.25 mM, pH8, Dako) or citrate buffer (0.01 M, pH6, SIGMA-ALDRICH) in water bath at 98 °C for 20 min or pressure cooker. Endogenous peroxidases were blocked by incubation with 3% H2O2 for 15 min at room temperature, followed by incubation for 30 min with 2% BSA to block non-specific binding. The sections were then incubated with primary antibodies anti-human CD68 (Dako, KP-1 clone, diluted 1:1000), CD20 (Dako, L26 clone, diluted 1:200), CD8 (Dako, C8/144B clone, diluted 1:100), PD-1 (Abcam, NAT105 clone, diluted 1:50), CD45RO (Dako, UCHL1 clone, diluted 1:200), IL-17 (R&D, AF-317-NA, 1:500) for 1 h at room temperature, followed by incubation with the detection system MACH 1 (Biocare Medical) or Anti-Goat Polymer kit (Biocare Medical). Diaminobenzidine tetrahydrochloride (Biocare Medical) was used as chromogen. Nuclei were lightly counterstained with a freshly made hematoxylin solution (Dako). The sections were then washed in water, mounted and analysed under an optical microscopy.
3
Oil Red O Staining for Lipid Droplets
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For Oil Red O staining, we used air-dried and thawed frozen sections or cultured hepatocytes. Respective samples were fixed in 4% paraformaldehyde solution for 20 min and rinsed three times in deionized water to remove excess formaldehyde. Thereafter, the sections were stained in Oil Red O (Sigma-Aldrich) solution, for 30 min at room temperature. Finally, the sections were extensively washed in deionized water, counterstained in hematoxylin solution (DAKO), rinsed with running tap water for 10 min, and mounted in aqueous mounting solution (PermaFluor, Thermo Scientific, Bonn, Germany).
4
Immunohistochemical Analysis of Tissue Sections
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The sectioned slides were passed through a series of xylene and ethanol solutions (100%, 90%, 80%, 70%) to remove the paraffin and then hydrated with distilled water and PBS. In order to reduce nonspecific binding, the slides were incubated with normal serum. The blocked slides were incubated with primary antibody (Table S2 ) for 15 h at 4 °C and 1 h at room temperature. The slides were then rinsed with PBS and incubated with biotinylated secondary antibodies (Vector Laboratories) for 1 h at room temperature. The slides were again rinsed with PBS and then incubated with ABC reagent (Vector Laboratories) according to the manufacturer’s instructions. After washing with PBS, the slides were developed for 5 min using a 3,3′-diaminobenzidine tetrahydrochloride hydrate (DAB; Sigma-Aldrich). Then, the slides were washed with PBS, followed by distilled water, and counterstained with hematoxylin solution (DAKO, Glostrup Kommune, Denmark). After the slides were washed, they were dehydrated with absolute alcohol and mounted using xylene and dibutyl phthalate in xylene (DPX; Sigma-Aldrich). Images of the stained slides were acquired with an optical microscope (Olympus Optical Co., Tokyo, Japan), and the intensity was analyzed using ImageJ software (NIH, Bethesda, MD, USA).
5
Immunohistochemical Analysis of Epithelial Markers
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Immunohistochemistry (IHC) was done on paraffin‐embedded tissue with Mucin 1 (MUC1), epithelial cell adhesion molecule (EpCAM), and epidermal growth factor receptor (EGFR) using the standard immunohistochemistry technique. IHC staining was done manually after antigen retrieval by boiling slides in 10 mm sodium citrate buffered distilled water (pH 6.0) for 20 min in a 97 °C water bath, followed by a 30 min cooldown period. Primary antibodies used were monoclonal anti‐MUC1 antibody (catalog no. 10‐M93B) and anti‐EpCAM antibody (catalog no. ab20160) at a dilution of 1:200 and anti‐EGFR antibody (catalog no. sc365829) at a dilution of 1:100. Primary antibodies were incubated for 19 h at 4 °C. The Dako REAL Peroxidase Detection System Kit was used according to the manufacturer's instructions, including the ready‐to‐use‐anti‐rabbit/mouse secondary antibody (catalog no. K5007) and counterstained with hematoxylin solution (catalog no. 03 971)
6
Immunohistochemical Analysis of CD68, CD163, and LXRα in Formalin-Fixed Tissues
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2-μm-thick consecutive tissue sections were prepared from formalin-fixed and paraffin-embedded tissues, provided by the Pathology Department of the Humanitas Clinical and Research Center, and processed for immunohistochemistry. Briefly, after deparaffinization and rehydration, antigen retrieval was performed by heat treatment using EDTA buffer (Dako; 0.25 mM, pH 8) in water bath at 98°C for 20 min. Endogenous peroxidases were blocked by incubation with Peroxidase-Blocking Solution for 15 min at room temperature, followed by incubation for 20 min with Background Sniper (Biocare Medical) to block nonspecific binding. The sections were then incubated with primary antibodies anti-human CD68 (Dako; KP-1 clone, diluted 1:1,000), CD163 (Leica Biosystems, 10D6 clone, diluted 1:200) for 1 h at room temperature, followed by incubation with the detection system EnVision+System HRP-labeled anti-mouse (Dako). Diaminobenzidine tetrahydrochloride (Biocare Medical) was used as chromogen. Nuclei were lightly counterstained with a freshly made hematoxylin solution (Dako). LXR staining was performed with anti-human LXRα antibody (LSBio; polyclonal, diluted 1:100), together with anti-CD163, followed by incubation with the detection system Mach2 Double Stain 1 (Biocare Medical). Ferangi Blue (Biocare Medical) and 3,3′-Diaminobenzidine were used as chromogens.
7
Immunohistochemistry of Human Breast Tissues
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Human breast tissues, provided by Seoul National University Hospital with approval from the SNUH Institutional Review Board (IRB; IRB No. 1904–141-1029), were fixed in 4% paraformaldehyde for 24 h at 4 °C and embedded in paraffin. The paraffin blocks were cut into 4-μm-thick sections, and the paraffin slides were deparaffinized with xylene and hydrated with graded ethanol. Antigen was unmasked by boiling the sections in 100 mM citrate buffer (pH 6.0) for 10 min and endogenous peroxidase activity was blocked with 3% hydrogen peroxide for 10 min. The slides were blocked in 5% normal goat serum (Vector Laboratories, Burlingame, CA, USA) for 1 h and incubated with primary antibodies (diluted 1:100–200 in antibody diluent solution; Life Technologies, Carlsbad, CA, USA) overnight at 4 °C. The following day, sections were incubated with biotin-conjugated secondary antibodies (diluted 1:100–200 in antibody diluent solution; Vector Laboratories) for 1 h and incubated with avidin-biotin complex reagent for 1 h. The slides were visualized using DAB staining solution (Dako, Santa Clara, CA, USA) followed by counterstaining with hematoxylin solution (Dako). Images of stained sections were obtained using the LAS microscope (Leica Microsystem, Wetzlar, Germany).
8
Immunohistochemical Analysis of CHI3L1
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The expression of CHI3L1 was evaluated by IHC. All specimens were sliced into a thickness of 4 μm, dewaxed and hydrated. Tris-EDTA solution (pH 9.0) was added and heated for antigen retrieval. After cooling to room temperature, a CHI3L1 antibody (Dilution, 1: 50; bs-10215R, Bioss Antibody, Boston, MA, USA) was added, and the sections were incubated overnight at 4℃. The slides were washed three times with PBS for 15 minutes, and then incubated with biotin-labeled goat anti-rabbit IgG secondary antibody (Dilution, 1:200; Vector laboratories, Burlingame, CA, USA) for 1 hour at RT. After washing the slides, the avidin-biotin-labeled enzyme complex (Vector) was reacted at RT for 45 minutes. AEC3-amino-9-ethylcarbazole (Abcam, Cambridge, MA, USA) was reacted in an incubator at 37℃ for 8 minutes. Finally, it was immersed in a hematoxylin solution (Dako, Glostrup, Denmark) for 1 minute to induce a reaction. For the negative control, PBS was used instead of the primary antibody. The stained slides were captured with an upright microscope (Axio imager2: ZEISS, Berlin, Germany) (magnification: 400 times).
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