As previously described (Lu et al., 2015 (link)), the tissue sections were dewaxed and washed three times with PBS. For non-specific inhibition, each section was incubated in 10% normal goat serum (Boster, Wuhan, China) for 30 min at 37°C and then with primary antibody, HIF-1α (Abcam, USA), overnight at 4°C. The sections were washed three times with PBS and were incubated with secondary antibody linked with biotin, and then with HRP-marked anti-biotin (Boster) for 30 min at 37°C. Subsequently, the sections were incubated in freshly prepared diaminobenzidine (Boster) and subsequently counterstained with hematoxylin (Boster). The sections were then observed under an optical microscope (200×) (Olympus, Tokyo, Japan).
Hematoxylin
Manufactured by Boster Bio
Sourced in China
Hematoxylin is a versatile staining agent used in various laboratory applications. It is a natural dye extracted from the heartwood of the Logwood tree (Haematoxylum campechianum). Hematoxylin is primarily used in histology and cytology to stain biological samples, highlighting cell nuclei and other cellular structures. Its core function is to provide a contrast-enhancing stain, enabling the visualization and differentiation of tissues and cells under a microscope.
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Lab products found in correlation
Dab solution, by CWBIO (2 mentions)
Hif 1α, by Abcam (1 mentions)
Normal goat serum (ngs), by Boster Bio (1 mentions)
Diaminobenzidine dab kit, by Boster Bio (1 mentions)
Bx40 light microscope, by Olympus (1 mentions)
3 3 diaminobenzidine (dab), by Agilent Technologies (1 mentions)
Horseradish peroxidase conjugated goat anti rabbit secondary antibody, by Abcam (1 mentions)
Anti ihh, by Abcam (1 mentions)
Hematoxylin, by CWBIO (1 mentions)
3 3 diaminobenzidine (dab), by Boster Bio (1 mentions)
Ix71 inverted light microscope, by Olympus (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Cell Signaling Technology (1 mentions)
Image pro plus 6, by Media Cybernetics (1 mentions)
Goat anti rabbit igg, by Boster Bio (1 mentions)
Axiovert 100m, by Zeiss (1 mentions)
Avidin biotin blocking buffer, by Vector Laboratories (1 mentions)
Dab reagent, by ZSGB-BIO (1 mentions)
Optical microscope, by Olympus (1 mentions)
16 protocols using hematoxylin
1
Immunohistochemical Analysis of HIF-1α Expression
2
IGF2BP1 Immunohistochemistry Analysis
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Formalin-fixed and paraffin-embedded tissue sections were deparaffinized in xylene and rehydrated in a graded series of alcohol solutions, followed by antigen retrieval and blockage with 3% bovine serum albumin for 30 min. Tissue sections were incubated with primary antibodies at optimal concentrations overnight at 4 °C. Then, the biotinylated sections were incubated with the secondary antibody (Boster, Wuhan, China) for 1 h at room temperature. Finally, the sections were stained with a diaminobenzidine (DAB) kit (Boster, Wuhan, China) and counterstained with hematoxylin (Boster, Wuhan, China). Staining was independently assessed by two experienced pathologists at the The Affiliated Cancer Hospital of Zhengzhou University. Images were obtained using a microscope (Olympus, Tokyo, Japan). IGF2BP1 staining intensity was classified as 0 (negative), 1 (weak), 2 (moderate), or 3 (strong). The staining proportion was quantified as 0 (negative), 1 (0.01%–50%), and 2 (51%–100%). The staining score of each sample was calculated as the proportional score × intensity score. Patients were grouped as low IGF2BP1 expression when the staining score was ≤ 2, and as high IGF2BP1 expression when the score was ≥ 3.
3
Immunohistochemical Analysis of Immune Markers
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For immunohistochemistry, sections were dipped in 10 mM citrate buffer (pH 6.0) and heated in a microwave oven for 15 min to thermally induce antigen epitope retrieval. After pretreatment with 3% H2O2 in PBS for 15 min to inactivate endogenous peroxidase activity and blocking with 5% BSA at room temperature, the sections were incubated with antibodies (Abcam, MA, USA) against CD68, CD11b, CD86, or CD163. Sections were washed extensively with PBS, incubated with relevant secondary antibodies, washed again, stained with 3,3′-diaminobenzidine (Dako, CA, USA), and finally counterstained with hematoxylin (Boster, Wuhan, China).
All images were captured from five consecutive microscopic fields using an Olympus BX40 light microscope (Olympus, Japan) coupled to a Dinolite AM423X microcamera (AmMo Electronics Corp, Taiwan) at 200 × magnification and processed with Image-Pro Plus 6.0 software.
All images were captured from five consecutive microscopic fields using an Olympus BX40 light microscope (Olympus, Japan) coupled to a Dinolite AM423X microcamera (AmMo Electronics Corp, Taiwan) at 200 × magnification and processed with Image-Pro Plus 6.0 software.
4
Quantifying GRHL2 Expression in Bladder Tissues
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The expression of GRHL2 in bladder tissues was evaluated by immunohistochemistry assay. In brief, the fresh bladder cancer tissues and the adjacent normal tissues were fixed in 10% natural formalin, embedded in paraffin and sliced into sections of 5 μm in thickness. After dewaxing and antigen retrieval, the paraffin-embedded tissue sections were incubated with rabbit anti-Human GRHL2 primary antibody (1:1000, abcam, Cambridge, MA, USA) at 4°C overnight according to manufacturer’s protocols. Sections were then washed and incubated with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (1:200, abcam, Cambridge, MA, USA) for 2 h at room temperature. Diaminobenzidine (DAB) plus kit (TIANGEN, Beijing, China) was used for visualization. Hematoxylin (BOSTER, Wuhan, China) was then applied for counter-staining. The intensity of DAB immunostaining was quantified using Image J software (NIH, Bethesda, MD).
5
Immunohistochemical Analysis of IHH Signaling
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The cartilage sections were deparaffinized, rehydrated, antigen repaired, and were incubated with an IHH antibody at the dilution of 1:50 (Abcam) overnight. The primary antibodies for the protein detection in rat tissue sections included anti-IHH (1:200; Abcam), anti-Gli1 (1:200; Abways), anti-Runx2 (1:200; CST), anti-Mmp13 (1:50; Boiss) and anti-Col2a1 (1:50; Boster). The protein samples were stained using the Diaminobenzidine (DAB) substrate kit and counterstained with hematoxylin (Boster). Image J software was used to analyze the mean optical density of IHH staining.
6
Immunohistochemical Localization of StAR3 Protein
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The technique of immunohistochemical staining was used to determine the localization of the StAR3 protein. Intestine and adductor tissues were dissected from golden and brown scallops and washed by PBS buffer. These samples were fixed in 10 % formalin, dehydrated, cleared, embedded in paraffin and cut to sections of 3 μm thickness before staining with hematoxylin (Boster, Wuhan, China). After 10 min treatment with 3 % H2O2 solution, tissue sections were washed in PBS and blocked with 5 % BSA for 30 min in a humidified box at room temperature, and then incubated with anti-rStAR3 rabbit antibody (diluted 1: 250) overnight at 4 °C. Then, the sections were washed with PBS and incubated with the horseradish peroxidase-labeled anti-rabbit IgG goat antibody (diluted 1: 500) (Boster, Wuhan, China) at room temperature for 30 min. After washing three times in PBS, the sections were developed with DAB solution (CWBIO, Beijing, China) for 10 min and counterstained in hematoxylin for 3 min. Photomicrographs were taken (EVOS Cell Imaging Systems).
7
Immunohistochemistry Staining of NK Cells
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Protocol used for IHC staining was described in our previous research.21 (link) Following antibodies were used to examine NK cell infiltration in mice tumors: CD56 (1:1000, clone 1E8C9, Cat# 60238-1-Ig, Proteintech) and goat anti-mouse IgG (HRP-linked) (1:2000, Cat# AP124P, Merck Millipore). After antibody incubation, slices were stained with stained with DAB (Cat# AR1022, BOSTER, Wuhan, China), and hematoxylin (Cat# AR0005, BOSTER) was used to counterstain the nucleus. Images of IHC staining of tissue slices were captured using an IX71 inverted light microscope (Olympus, Tokyo, Japan).
8
Immunohistochemical Analysis of Antioxidant Proteins
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Tissue sections were prepared as per the method used above. After dewaxing, the sections were incubated in a water bath kettle oven at 98°C for 15 min with target retrieval solution for antigen retrieval, followed by treatment with 3% EDTA for 15 min and 5% BSA for 30 min. The sections were incubated with primary antibodies (Nrf2: 1 : 400, Cell Signaling Technology; HO-1: 1 : 400, Cell Signaling Technology) at 4°C overnight, washed with PBS, and incubated with horseradish peroxidase-conjugated secondary antibody (1 : 400, Cell Signaling Technology) for 1.5 h at room temperature. For color development, sections were treated with the peroxidase substrate, DAB, and counterstained with hematoxylin (Boster Biological Technology Co. Ltd.).
9
Quantifying Tight Junction Protein Expression
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The expressions of proliferating cell nuclear antigen (PCNA) and tight junction proteins as ZO-1, occludin, claudin-3, and claudin-1 were determined using immunohistochemical analysis as described by [25 (link)].
The stained sections were incubated with the primary antibodies described by [25 (link)]: anti-PCNA (1 : 200; Changsha Kainuo Biological Technology Co., Ltd., Changsha, China), ZO-1 polyclonal antibody (1 : 100; Abcam, Cambridge, UK), occludin polyclonal antibody (1 : 100; Abcam), claudin-3 poly-clonal antibody (1 : 100; Abcam), claudin-1 polyclonal antibody (1 : 100; Abcam), E-claudin poly-clonal antibody (1 : 100; Abcam), Rabbit hypersensitivity 2-step immunohistochemical kit (Boster Biological Technology), diaminobenzidine (Boster Biological Technology), and hematoxylin (Boster Biological Technology). The PCNA labeling index was expressed as the ratio of cells that were positively stained for PCNA to all epithelial cells. The expressions of tight junction proteins were expressed as the average optical density (the ratio of integrated optical density to the area of tissue) in at least 5 areas. All areas were randomly selected for counting at less than 200-fold magnification and all data expressed as the relative values to those of control piglets.
The stained sections were incubated with the primary antibodies described by [25 (link)]: anti-PCNA (1 : 200; Changsha Kainuo Biological Technology Co., Ltd., Changsha, China), ZO-1 polyclonal antibody (1 : 100; Abcam, Cambridge, UK), occludin polyclonal antibody (1 : 100; Abcam), claudin-3 poly-clonal antibody (1 : 100; Abcam), claudin-1 polyclonal antibody (1 : 100; Abcam), E-claudin poly-clonal antibody (1 : 100; Abcam), Rabbit hypersensitivity 2-step immunohistochemical kit (Boster Biological Technology), diaminobenzidine (Boster Biological Technology), and hematoxylin (Boster Biological Technology). The PCNA labeling index was expressed as the ratio of cells that were positively stained for PCNA to all epithelial cells. The expressions of tight junction proteins were expressed as the average optical density (the ratio of integrated optical density to the area of tissue) in at least 5 areas. All areas were randomly selected for counting at less than 200-fold magnification and all data expressed as the relative values to those of control piglets.
10
Immunohistochemical Analysis of Lung Tissues
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After heat fixed, deparaffinized, and rehydrated through graded alcohols to distilled water, lung tissue paraffin sections (4 μm) were blocked with 5% bull serum albumin and incubated with the primary antibodies against Mucin (MUC) 5ac, MUC5b, transforming growth factor- (TGF-) β1, α-smooth muscle-actin (SMA) (Bioss, Beijing, China), and Collagen I, Collagen III (Proteintech, Wuhan, China) overnight at 4°C. Subsequently, the sections were incubated with goat anti-rabbit IgG at 37°C for 30 minutes and counterstained by hematoxylin according to the manufacturers' instructions (Boster, Wuhan, China). Finally, images were captured and analyzed using the Image-ProPlus 6.0 software (Media Cybernetics, MD, USA).
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