Tissue paraffin sections were immersed in xylene for dewaxing and then successively placed in ethanol at a gradient concentration for hydration. Antigen was retrieved using 0.01M citrate buffer (pH 6.0) at 100°C for 10 min. Endogenous peroxidase was devitalized using 100 µl 3% H2O2 at room temperature for 10 min. Three percent BSA was used to block tissue section at room temperature for 1 hour. The primary antibody CYP2C8 (Abcam, USA) and Ki67 (Proteintech, USA) were respectively diluted according to the manufacturer’s instructions, and the sections were incubated overnight in primary antibody diluent at 4 °C. After washing thrice within PBS, the sections were incubated with corresponding secondary antibodies (ZSGB-Bio, China) at room temperature for 30 min. After washing twice in PBS to get rid of residual secondary antibodies, the tissue sections were dripped with an appropriate amount of the detection system V9000 (ZSGB-Bio, China) and incubated at 37°C for 20 min. After washing twice in PBS, the tissue sections were dripped with freshly prepared DAB solution (ZSGB-Bio, China) and incubated at room temperature for 5–10 min. When showing positive stain, the tissue sections were immediately washed to stop the chromogenic reaction. Then, the sections were counterstained with hematoxylin solution.
Dab solution
Manufactured by ZSGB-BIO
Sourced in China
The DAB solution is a versatile laboratory reagent used in various immunohistochemical and immunocytochemical techniques. It serves as a chromogenic substrate, providing a brown staining reaction when catalyzed by the enzyme horseradish peroxidase (HRP). This solution is a core component in the visualization and detection of target proteins or antigens in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Purelink rna mini kit, by Thermo Fisher Scientific (2 mentions)
Cyp2c8, by Abcam (1 mentions)
One step primescript rt pcr kit, by Takara Bio (1 mentions)
Capsaicin, by Merck Group (1 mentions)
Hydroxyproline assay kit, by Nanjing Jiancheng (1 mentions)
Goat serum, by ZSGB-BIO (1 mentions)
Edta buffer, by ZSGB-BIO (1 mentions)
Anti cbx4, by Abcam (1 mentions)
Hematoxylin, by ZSGB-BIO (1 mentions)
Horseradish peroxidase conjugated streptavidin, by Thermo Fisher Scientific (1 mentions)
Ix81 digital camera, by Olympus (1 mentions)
Uplanfl n digital camera, by Olympus (1 mentions)
Fv1000, by Olympus (1 mentions)
Bx51 microscope, by Olympus (1 mentions)
Goat serum, by Vector Laboratories (1 mentions)
Image pro plus 6, by Media Cybernetics (1 mentions)
Zli 9021, by ZSGB-BIO (1 mentions)
Ds ri1, by Nikon (1 mentions)
Eclipse 80i microscope, by Nikon (1 mentions)
Goat anti rabbit secondary antibody, by ZSGB-BIO (1 mentions)
11 protocols using dab solution
1
Immunohistochemical Analysis of CYP2C8 and Ki67
2
Bleomycin-Induced Lung Fibrosis in Guinea Pigs
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Specific pathogen-free male Dunkin–Hartley guinea pigs (250~300 g) were purchased from Vital River Laboratories (Beijing, China). Bleomycin (BLM) was purchased from Hisun-Pfizer Pharmaceuticals (Zhejiang, China). Capsaicin was purchased from Sigma Aldrich (Seattle, USA). PureLink™ RNA Mini Kit was purchased from Thermo Fisher Scientific (Carlsbad, USA), One-Step PrimeScript™ RT-PCR Kit from TaKaRa (Dalian, China). Primers were synthesized by Sangon (Shanghai, China). TRPV1 primary rabbit antibody was purchased from Abcam (Cambridge, MA, USA) and TRPA1 primary rabbit antibody from Novus-Bio (Littleton, USA). The poly-HRP anti-rabbit IgG secondary antibody and DAB solution were purchased from ZSGB-Bio (Beijing, China). Hydroxyproline assay kit was purchased from Jiancheng Bioengineering Institute (Jiangsu, China).
3
Immunodetection of Insulin in Pancreatic Tissue
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Sections were cut from embedded pancreatic slices and deparaffinized in dimethyl benzene. The immunohistochemistry procedures were set up according to the manufacturer’s instructions (Zsbio, Beijing, China). Sections were blocked using 3% hydrogen peroxide and 10% serum and then incubated with primary antibody (anti-insulin, Bioss, Beijing, China). Avidin anti-rabbit antibody served as the secondary antibody followed by horseradish peroxidase anti-avidin antibody. Horseradish peroxidase activity was detected using a DAB solution (Zsbio, Beijing, China). Finally, the reaction was stopped, and sections were counterstained with hematoxylin.
4
Immunohistochemical Analysis of CBX4 in Human Disc Tissue
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Human NP tissue was obtained from patients who underwent lumbar interbody fusion for degenerative disc diseases, and the procedures were approved by the Medical Ethics Committee of Sun Yat-sen Memorial Hospital, Sun Yat-sen University. From September 2020 to March 2021, 10 disc samples (male:female=3:7) were collected, evaluated by Pfirrmann classification and classified into 2 groups.
Human tissues paraffin sections were deparaffinized with xylene and rehydrated, followed by retrieval with EDTA buffer (ZSGB-BIO, Beijing, China) for 10 min at 100°C. Hydrogen peroxide (3%) was used to quench endogenous peroxidase activity. Then, goat serum (ZSGB-BIO) was used to block the paraffin sections for 30 min. The paraffin sections were incubated with PBS or anti-CBX4 (1:100; Abcam, Cambridge, UK) primary antibodies overnight at 4°C. After being incubated with biotin-labeled secondary antibodies for 30 min at room temperature and HRP-conjugated streptavidin for 20 min, the sections were developed with DAB solution (ZSGB-BIO), and counterstained with 1% hematoxylin. Lastly, sections were mounted, photographed under a microscope (Nikon, Tokyo, Japan).
Human tissues paraffin sections were deparaffinized with xylene and rehydrated, followed by retrieval with EDTA buffer (ZSGB-BIO, Beijing, China) for 10 min at 100°C. Hydrogen peroxide (3%) was used to quench endogenous peroxidase activity. Then, goat serum (ZSGB-BIO) was used to block the paraffin sections for 30 min. The paraffin sections were incubated with PBS or anti-CBX4 (1:100; Abcam, Cambridge, UK) primary antibodies overnight at 4°C. After being incubated with biotin-labeled secondary antibodies for 30 min at room temperature and HRP-conjugated streptavidin for 20 min, the sections were developed with DAB solution (ZSGB-BIO), and counterstained with 1% hematoxylin. Lastly, sections were mounted, photographed under a microscope (Nikon, Tokyo, Japan).
5
Immunohistochemical and Immunofluorescence Analyses of FXYD6 and Na+/K+-ATPase
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For DAB staining, the commercial human tissue array was deparaffinized and stained firstly with FD10 (1 μg/mL), then incubated with Biotin-conjugated anti-mouse secondary antibody (ZSGB-BIO) and horseradish peroxidase-conjugated streptavidin (Thermo Fisher). The binding was detected by DAB solution (ZSGB-BIO). The tissues were then counter stained using hematoxylin (ZSGB-BIO). Images were taken with an OLYMPUS BX51 microscope with an UPlanFL N digital camera.
For immunohisto-fluorescence, the commercial human tissue array (AOMEI) was deparaffinized and co-stained with both anti-FXYD6 mAb FD10 (1 μg/mL) and anti-Na+/K+-ATPase α1 subunit antibody, followed by fluorescent-labeled secondary antibody. Nuclei were stained with DAPI. Pictures were taken with a confocal laser scanning microscope (Olympus FV1000) with an Olympus IX81 digital camera.
For immunohisto-fluorescence, the commercial human tissue array (AOMEI) was deparaffinized and co-stained with both anti-FXYD6 mAb FD10 (1 μg/mL) and anti-Na+/K+-ATPase α1 subunit antibody, followed by fluorescent-labeled secondary antibody. Nuclei were stained with DAPI. Pictures were taken with a confocal laser scanning microscope (Olympus FV1000) with an Olympus IX81 digital camera.
6
Immunohistochemical Analysis of Kidney Samples
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After dewaxing and rehydration, slides were incubated with 3% H2O2 for quenching endogenous peroxidase activity and then heated in boiling antigen unmasking solution for antigen retrieval. Slides were blocked with 5% goat serum (Vector Laboratories, UK) for 1 h, then incubated with primary antibody in a humidified chamber at 4℃ overnight. Positive signal was visualized by DAB solution (ZSGB-BIO, Beijing, China). The images were visualized and acquired using the Leica DM 1000 microscope image system. Quantitative evaluation involved the use of Image-Pro Plus 6.0 (Media Cybernetics, Rockville, MD, USA) 58 (link). Positive signals and the number of positive cells in the kidney were quantified in 10 bright fields (×200) in the eyepiece of the microscope.
7
Immunohistochemical Analysis of Rat Arteries
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Following the standard procedure, paraffin‐embedded rat artery sections were rehydrated by dimethylbenzene and gradient ethanol. Then, 0.05 mol/L sodium citrate buffer (pH 6.0) was introduced for heat‐mediated antigen retrieval. Slides were submerged in 3% hydrogen peroxide for 10 minutes to remove endogenous peroxidase. After a wash step, the slides were blocked with 10% goat serum (ZLI‐9021; ZSGB‐BIO) for 30 minutes at 37°C, followed by an overnight incubation with primary antibodies against α‐SMA (1:500 dilution), calponin 1 (1:200 dilution) and RUNX2 (1:100 dilution) at 4°C in a humid box. After 30 minutes of incubation with the appropriate secondary antibody at 37°C, the slides were reacted with DAB solution (ZSGB‐BIO). Haematoxylin was applied to counterstain the nucleus. The tissue sections were visualized under a Nikon Eclipse 80i microscope equipped with a digital camera (DS‐Ri1; Nikon) and were analysed with Image‐Pro Plus 6.0 software.
8
Immunohistochemical Analysis of Colorectal Cancer
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Paraffin-embedded human CRC dissected tissues were baked in an oven at 70 °C for 2 h. The sections were then dewaxed in the xylene solution, rehydrated in a decreasing concentration of ethanol, and rinsed under running water. The antigens were unmasked in a pressure cooker with the citrate repair solution (ZSGB-bio, Beijing, China) at 2100 W for 30 s, followed by adjusting to 800 W for 10 min and cooling to room temperature. The endogenous peroxidases were blocked by the 3% hydrogen peroxide solution for 15 min away from light. The sections were then incubated with primary antibodies at 4 °C overnight and incubated with a goat anti-rabbit secondary antibody (ZSGB-bio) for 30 min at 37 °C. DAB solution (ZSGB-bio) was used for color development. After hematoxylin staining, the sections were dehydrated in an increasing gradient of ethanol and xylene. Two experienced pathologists independently observed and scored the degree of staining in the sections. The staining intensity was scored as follows: 0 = no staining, 1 = light yellow, 2 = brownish yellow, and 3 = brown.
9
Immunohistochemical Analysis of Joint Tissues
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The left joint specimen sections were processed as described for the immunofluorescence assay up to the incubation step with the primary antibody. The antibody dilution ratio for COL-2 was 1 : 200, and the ratio for MMP-3 was 1 : 500 (Abcam, Cambridge, UK). The sections were then incubated with a biotin-labeled anti-rabbit secondary antibody (ZSGB-BIO, Beijing, China), stained with 3,3′-diaminobenzidine (DAB) solution (ZSGB-BIO, Beijing, China), counterstained with hematoxylin to visualize the nuclei, and sealed with neutral gum. Image-Pro Plus 6.0 software was used to calculate the AOD score of the positive product.
10
Inhibition of NAT10 Suppresses Tumor Growth
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Before all animal studies, ethics approval was obtained from the Animal Care Committee of Changhai Hospital, Second Military Medical University (Naval Medical University). For xenograft experiments, a total of 20 male C57/BL6 mice were purchased from the Model Animal Research Center (Nanjing, China). All mice were randomly and evenly divided into four groups. AKR cells stably transfected with sh-NC or sh-NAT10#1 were inoculated subcutaneously on the right dorsal side of mice at a density of 2 × 106 cells per mouse. Two groups of mice underwent PD-1 treatment at 7 days after injection. Tumor volume was recorded and calculated every four days according to the formula (length × width2/2). At day 28, mice were sacrificed, and each tumor was isolated and weighed. Tumor samples from the four different groups were collected for IHC staining.
For IHC staining, after the sections were deparaffinized and rehydrated, the specimens were incubated in EDTA buffer (1 mM, PH 8.0) for antigen retrieval using a high-pressure method. Then, tissue sections were incubated overnight at 4 °C with primary antibodies, including anti-NAT10, anti-CD163, and anti-CD86. DAB solution (ZSGB-BIO, Beijing, China) was used to detect target proteins, which were conjugated with a peroxidase enzyme to form a brown precipitate.
For IHC staining, after the sections were deparaffinized and rehydrated, the specimens were incubated in EDTA buffer (1 mM, PH 8.0) for antigen retrieval using a high-pressure method. Then, tissue sections were incubated overnight at 4 °C with primary antibodies, including anti-NAT10, anti-CD163, and anti-CD86. DAB solution (ZSGB-BIO, Beijing, China) was used to detect target proteins, which were conjugated with a peroxidase enzyme to form a brown precipitate.
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