Immunohistochemistry (IHC) staining was performed as described previously (Jiang et al., 2016 (link); Yi et al., 2021 (link)). In brief, the paraffin slices were soaked in xylene, 100% ethanol, 95% ethanol and 70% ethanol successively for dewaxing and hydration. After that, the slides were put into EDTA buffer (pH 9.0, MVS-0099, MXB Biotechnologies) and maintained at 100°C for 20 min to retrieve the antigen. The slides were blocked with blocking buffer (5% bovine serum albumin, FA016-100G, Genview) for 30 min at 37°C after treatment with 3% hydrogen peroxide for 40 min. The IRF9 primary antibody (Proteintech, 14167-1-AP, at 1:500 dilution) was added to the slides for incubation overnight at 4°C after removing the blocking solution. The peroxidase-conjugated secondary antibody (Kit-9902, MXB Biotechnologies) was incubated for 30 min at 37°C. A DAB kit (DAB-0031, MXB Biotechnologies) was used to develop the color, and the sections were counterstained with hematoxylin. The slices were mounted with neutral resins (10004160, Sinopharm) after dehydration and observed under the microscope. The intensity of medial IRF9 staining was quantified relative to the area of the medial layer to evaluate the relative expression level of IRF9.
Dab 0031
Manufactured by Beijing Strong Biotechnologies
Sourced in China
The DAB-0031 is a laboratory equipment product developed by Beijing Strong Biotechnologies. It is a device used for the detection and analysis of biological samples. The core function of the DAB-0031 is to provide accurate and reliable measurements of various analytes in a research or diagnostic setting.
Automatically generated - may contain errors
Lab products found in correlation
Kit 9710, by Beijing Strong Biotechnologies (3 mentions)
Kit 9720, by Beijing Strong Biotechnologies (2 mentions)
Mvs 0099, by Beijing Strong Biotechnologies (1 mentions)
Fa016 100g, by Genview (1 mentions)
Kit 9902, by Beijing Strong Biotechnologies (1 mentions)
Phase contrast microscope, by Leica (1 mentions)
Inverted phase contrast microscope, by Leica (1 mentions)
Hematoxylin, by Merck Group (1 mentions)
Kit 9707, by Beijing Strong Biotechnologies (1 mentions)
Caseviewer 2, by 3DHISTECH (1 mentions)
Aipathwell, by Wuhan Servicebio Technology (1 mentions)
Kit 7710, by Beijing Strong Biotechnologies (1 mentions)
Eclipse 90i, by Nikon (1 mentions)
Ab70287, by Abcam (1 mentions)
Ab78494, by Abcam (1 mentions)
S0001, by Affinity Biosciences (1 mentions)
Bs 1074r, by Bioss Antibodies (1 mentions)
Ab27278, by Abcam (1 mentions)
Ab33682, by Abcam (1 mentions)
10 protocols using dab 0031
1
Immunohistochemical Analysis of IRF9 Expression
2
Chondrocyte Morphology and Collagen II Identification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Chondrocyte morphology on different culture days and of different generations was observed under an inverted phase-contrast microscope (Leica Microsystems, Inc., Wetzlar, Germany) and images were captured. Second-generation chondrocytes are often selected for experimentation (27 (link)); therefore, type II collagen immunohistochemistry was applied to identify passage 2 chondrocytes. A total of 5×104 second-generation chondrocytes per well were implanted onto a sterile round coverglass in a 6-well plate. Chondrocytes in the 6-well plate (2 ml medium/well) were incubated for 48 h and were then randomly divided into two groups. The positive group was treated with 100 µl rabbit polyclonal antibody against collagen II (dilution 1:200; cat. no. ab34712; Abcam, Cambridge, UK), whereas the negative group was treated with 100 µl PBS. Both groups were incubated overnight at 4°C. After incubation, the two groups of chondrocytes were treated with a secondary antibody (cat. no. KIT-9707; MXB Biotechnologies, Inc., Fujian, China) at 37°C for 1 h, in accordance with the manufacturer's instructions; color was developed using a DAB kit (cat. no. DAB-0031; MXB Biotechnologies, Inc.); and the cells were stained with hematoxylin (Sigma-Aldrich; Merck KGaA) for 1 min. The staining of the two groups of cells was observed and compared under a phase-contrast microscope (Leica Microsystems, Inc.).
3
Renal Nephrin Expression Quantification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immunohistochemical staining was performed on 4% paraformaldehyde-fixed, paraffin-embedded 3-μm renal tissue sections. After antigen recovery, the sections were incubated with primary antibodies against nephrin (1:200, mouse, sc-377246) overnight at 4 °C, and then incubated with peroxidase-conjugated anti-mouse IgG antibodies. Reactions were stained with a DAB substrate kit (MXB biotechnologies, DAB-0031, Fuzhou, China), and counterstaining was performed using hematoxylin. The sections were captured under a microscope (Olympus, BX53, Tokyo, Japan), and Image ProPlus was used to quantify the average optical density value.
4
Immunohistochemical Staining Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immunohistochemical staining was performed using an immunohistochemistry kit (MXB, KIT-9710). To inhibit endogenous peroxidase, the tissue sections were first exposed to 1% H2O2 and then subjected to heat-induced antigen retrieval at 97°C for 15 min. After incubation with GPX4, ACSL4, MYH6, and MYH7 primary antibodies for 1 h, the sections were rinsed with phosphate-buffered solution and incubated again with the corresponding secondary antibodies. Finally, the sections were stained with 3,3-diaminobenzidine (DAB; MXB, DAB-0031) and counterstained with hematoxylin, followed by microscopic examination. The cells with brownish-yellow particles in their nuclei and/or cytoplasm were classified as immunoreactive cells.
5
Immunohistochemistry of Murine Colon Tissue
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Paraffin-embedded colonic sections of mice were deparaffinized using xylene, and rehydrated through graded ethanol. Immunohistochemistry kit (MXB Biotechnologies, KIT-9720) was used according to its manufacturer’s instructions for next processes. Briefly, 3% (vol/vol) H2O2 in methanol was firstly utilized to block endogenous peroxidase. Then sections immersed in citrate buffer were heated in microwave for 15 min to retrieve antigen. After cooling to room temperature, sections were blocked using goat serum and then incubated with primary antibodies overnight at 4 °C, followed by biotin-conjugated secondary antibodies incubation and subsequent streptavidin-HRP incubation. Immunoreactive tissues were visualized with DAB (MXB Biotechnologies, DAB-0031) as a chromogen and hematoxylin as counterstain.
6
Immunohistochemical Analysis of IL-6 Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
After dewaxing and dehydration, the antigens in the EDTA-treated samples were heat-repaired in an oven at 100 °C for 40 min. Three percent hydrogen peroxide (SP KIT-A2, MXB, Fuzhou, China) was used to block the sections for 10 min and in goat serum for 45 min (SP KIT-B1, MXB, Fuzhou, China). Sections were then incubated with IL-6 primary antibody (1:400; GXP263019, Genxspan, Alabama, USA) overnight at 4 °C for approximately 10 h. Slices were then washed with PBS (Hyclone, Cytiva, Marlborough, MA, USA) and incubated with a secondary antibody (KIT-5010, Maishin, Fuzhou, China) for 15 min at 37 °C. Nuclei were re-stained with hematoxylin (DAB0031, MXB, Fuzhou, China) for 30 s, dehydrated, and sealed in gum. For semi-quantitative analysis, the average optical density (AOD) of IL-6 in the stained tissues was calculated using Image J-Pro plus 6.0 (National Institutes of Health, Bethesda, MD). AOD = (integrated optical density)/area.
7
Immunohistochemical Analysis of Ki67 Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The deparaffinized and rehydrated tissue slide was pretreated for 25 min with peroxidase blocking buffer (CAT#LS-M24-100, LSBio, Seattle, WA, USA) at 28°C. Antigens were retrieved using citrate buffer (pH 6.0) at 28°C for 30 min. Afterwards, we inoculated the tissue slides for 1 hour with 5% BSA in PBS at 28°C. Subsequently, the slides were overnight inoculated with Anti-Ki67 antibody (CAT#9027T, Boston,MA, USA) at 4°C. Thereafter, the tissue chip was inoculated for 30 min with biotin-linked secondary antibody (CAT#KIT-7710, MXB Biotechnologies, Fuzhou, Fujian, China) at 28°C, followed by inoculation for 15 min with HRP-linked streptavidin (CAT#KIT-7710, MXB Biotechnologies) at 28°C. Finally, Ki67 signal was determined by DAB staining (CAT#DAB0031, MXB Biotechnologies). The images were captured with a panoramic scanner PANNORAMIC (3DHISTECH, Budapest, Hungary) using the software CaseViewer 2.4 (3DHISTECH). The integrated optical density (IOD) and Ki67+ area were analyzed with the software AIpathwell (Servicebio, Wuhan, China). Mean density of Ki67 was calculated using the formula: mean density = IOD/Ki67+ area.
8
Immunohistochemical Analysis of Kidney Injury Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mouse kidney tissue was fixed in 4% paraformaldehyde for 3 days and dehydrated using an alcohol gradient. The tissue was then immersed in an ethanol–xylene (1:1) mixture in xylene for 15–20 min until the tissue became transparent. Transparent tissue was placed in a mixture of paraffin and xylene (1:1) for 1 h, followed by paraffin embedding. Paraffin sections were cut into 5-μm-thick sections and dried in a 37°C incubator. The immunohistochemistry kit was purchased from MXB Biotechnologies (KIT-9710 and DAB-0031, China). Renal tissue paraffin slices were washed with water and subjected to antigen retrieval. The slices were then incubated with primary antibodies against Kim-1 (Abcam, 1:200, ab78494), NGAL (Abcam, 1:200, ab70287), and the appropriate biotin-conjugated secondary immunoglobulin G (1:1000, S0001, Affinity, USA). After mounting the slices, co-stained images were captured using a Nikon Eclipse 90i fluorescence microscope.
9
Immunohistochemical Analysis of Key Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
After the sections were deparaffinized and rehydrated, the experiments were performed following the IHC kit instructions (kit-9720, MXB, China) for MMP13 (18165-1-AP; 1:200; Proteintech), Nrf2 (bs-1074R; 1:500; Bioss), P53 (A0263; 1:100; Abclonal), SLC7A11 (DF12509; 1:100; Affinity Biosciences), Col2a1 (28459-1-AP; 1:1000; Proteintech), p-NF-κB p65 (bs-5662R; 1:200; Bioss), and GPX4 (67763-1-Ig; 1:2000; Proteintech). A series of primary antibodies were added to the sections of the four groups, and then all sections were incubated in a wet box at 4 °C overnight. The following day, the sections were incubated at room temperature for 1 h with a secondary antibody, and a DAB solution (dab-0031, MXB, China) was added for visualization. Finally, after neutral resin blocking, they were photographed under a microscope. Five fields were randomly selected for each sample, and the expression of each index was examined and averaged for statistical analysis.
10
Immunohistochemical Analysis of Lymphatic Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Three mice in each experimental group were randomly selected. After fixing the tumor tissues in 4% polymethylene formaldehyde for 24 h, they were cut into 4 μm sections. After the waxing was removed, the slides were treated accordingly using KIT-9710 (MXB Biotechnologies, Fuzhou, China). The slides were incubated with the primary antibodies against LYVE-1 (1:200, ab33682, Abcam), VEGF-C (1:100, CST2445, CST, Boston, MA, USA), or VEGFR-3 (1:100, ab27278, Abcam, Shanghai, China). After antibody incubation, slices were stained with DAB (DAB-0031, MXB Biotechnologies) and hematoxylin (G1140, Solaibao, Beijing, China). Finally, three fields were randomly selected under the optical microscope (400×), in which the brown-yellow particles were observed, then analyzed with Motic 6.0 image analysis system.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!