Kidney samples were fixed in 4% paraformaldehyde and embedded in paraffin. Sections that were 4-μm thick were cut for morphometric analysis and IHC. The sections were prepared for periodic acid-Schiff (PAS) and Sirius red staining, as described previously [32 ]. For immunohistological studies, immunoreactivity was detected in cortical sections with a specific primary antibody and an ABC staining kit and visualized with a DAB detection kit (both kits were obtained from Vector Laboratories Inc., Burlingame, CA, USA). Primary antibodies against collagen IV (1:200) and laminin (1:200) were obtained from Abcam. The sections were examined using an Olympus BX43F microscope. Linear measurements were obtained using an image analysis system (Image-Pro Plus 4.0, Media Cybernetics, Silver Spring, MD, USA).
Dab detection kit
Manufactured by Vector Laboratories
Sourced in United States
The DAB detection kit is a laboratory tool used to detect specific target molecules in tissue samples. It provides a simple and reliable method for visualizing proteins or other biomolecules of interest.
Automatically generated - may contain errors
Lab products found in correlation
Abc kit, by Vector Laboratories (5 mentions)
Eclipse ni microscope, by Nikon (2 mentions)
Deadend fluorometric tunel system, by Promega (2 mentions)
Charge coupled device camera, by Olympus (2 mentions)
Image pro plus 4, by Media Cybernetics (2 mentions)
Bx43f microscope, by Olympus (1 mentions)
Polymer hrp detection system, by ZSGB-BIO (1 mentions)
Goat serum, by Thermo Fisher Scientific (1 mentions)
H8070, by Solarbio (1 mentions)
Biotinylated goat anti mouse igg, by Vector Laboratories (1 mentions)
Biotinylated goat anti rabbit igg, by Vector Laboratories (1 mentions)
Anti olfm4, by Cell Signaling Technology (1 mentions)
Anti mmp 7, by Cell Signaling Technology (1 mentions)
Anti sox9, by Merck Group (1 mentions)
Vectastain kit, by Vector Laboratories (1 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
Dmi3000b, by Leica (1 mentions)
Anti ki67, by BD (1 mentions)
Avidin biotin blocking kit, by Vector Laboratories (1 mentions)
Target antigen retrieval solution, by Agilent Technologies (1 mentions)
21 protocols using dab detection kit
1
Histological Analysis of Kidney Samples
2
Immunohistochemical Analysis of Synaptopodin
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The fixed kidney section of a 4 μm thickness (Leica Company, Germany, RM2235) were deparaffinized in xylene 3333 rehydrated in a graded series of alcohols. Subsequently the sections were placed in 3% H2O2 for 10 min to eliminate endogenous peroxidase activity. After pepsin antigen retrieval for 30 min, the sections were washed with PBS 3 times for 3 min each time and then blocked with 2% BSA for 0.5 h at room temperature. The sections were incubated with mouse anti-synaptopodin antibody (1:200, santa cruz biotechnology, sc-515842) at 37°C for 2 h or 4°C overnight. The sections were stained using a polymer HRP detection system (ZSGB-BIO, Beijing, China) and visualized with a DAB detection kit (Vector Laboratories Inc., Burlingame, CA, United States). After conventional dewatering and neutral balsam mounting, photographs were blindly taken at random felds under an Olympus BX43F fuorescence microscope (OLYMPUS, Japan) (Chen et al., 2019 (link)).
3
Immunohistochemical Analysis of Xenograft Tumors
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Xenograft tumor samples were fixed in 10% formalin solution for 24 hours and transferred to 70% ethanol. Then tissues were embedded in paraffin wax according to embedding machine manufactures instructions. And 4-μm sections were prepared. Immunohistochemistry was performed according to standard protocols. Antigen retrieval was achieved by heating sections in 95 °C citrate buffer for 10 minutes. Sections were incubated with specific antibodies overnight at 4 °C. For CD31 (1:100) and Ki67 (1:100) staining, the dark brown signal was revealed after incubation with the ABC kit (Vector), followed by a diaminobenzidine (DAB) and hydrogen peroxide reaction using the DAB detection kit (Vector). Counterstaining was performed by incubating the slides in Hematoxylin for 5min. For α-SMA (1:200) staining, Alexa fluor 568-conjugated secondary antibody was used. The nuclei were visualized by incubation with DAPI, and images were examined with a fluorescent microscope. Appropriate controls were used in all cases by incubating sections with all except the primary antibodies. No staining was observed under these conditions.
4
Immunohistochemical Analysis of Tissue Markers
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Paraffin sections were routinely deparaffinized in xylene and rehydrated in decreased alcohol gradient. After a brief wash with deionized water, tissue antigen was retrieved through heating in sodium citrate buffer for 15 min, followed by incubation with 3% H2O2 for 10 min to quench the endogenous peroxidase. Following three times of PBS washing, non-specific antigen-binding sites were blocked with 10% goat serum in PBS (Gibco) overnight. The sections were then incubated with anti-Sox9 (1:5000, EMD Millipore, Cat# AB5535); anti-ki67(1:500, BD, Cat# 550609); anti-MMP-7 (1:100, Cell Signaling Technology, Cat# 3801S); anti-Olfm4 (1:500, Cell Signaling Technology, Cat# 39141S) at 4 °C overnight. After washing in PBS, the sections were incubated with biotinylated goat anti-rabbit IgG (1:1000, Vector Cat# BA-1000) or biotinylated goat anti-mouse IgG (1:1000, Vector Cat# BA-9200) for 2 hr. in room temperature. The detection was performed with a DAB detection kit (Vector Laboratories, Cat# SK-4100) and a VECTASTAIN kit (Vector Laboratories, Cat# PK-7100) according to the manufacturer’s instructions. Following counter-staining in Hematoxylin (Solarbio® LIFE SCIENCE Cat# H8070) and mounting with neutral balsam (Solarbio® LIFE SCIENCE Cat# 96949–21-2). Sections were observed using a light-field microscope (Leica DMI3000B).
5
Liver Tissue Immunostaining Protocol
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Mice were killed and 20–200 mg fresh liver samples were immediately snap frozen in tubes. Remaining samples were fixed with 4% paraformaldehyde overnight for paraffin blocks. Paraffin sections were used either for haematoxylin and eosin or immunostaining.
Sections were treated with Target Antigen Retrieval Solution (Dako). Endogenous peroxidase was blocked using 3% hydrogen peroxide followed by an overnight incubation with primary mouse anti-HPD antibody (Santa Cruz sc-271672) diluted 1:100. Biotinylated secondary antibody was incubated for 30 min. Detection of HPD was performed using the M.O.M Vectastain kit and DAB detection kit (Vector Laboratories) according to the manufacturer's recommendations. Haematoxylin was used for counterstaining.
For immunofluorescence detection, endogenous biotin was blocked using Avidin/Biotin blocking kit (Vector laboratories) before incubation with biotinylated secondary antibody. Streptavidin-AlexaFluor488 (Thermo Fisher Scientific, cat# S32354) was incubated for 30 min at a dilution of 1:1,000. EdU was detected using the Click-iT Plus EdU Alexa Fluor 594 Imaging Kit (Molecular Probes) according to the manufacturer's instructions.
Sections were treated with Target Antigen Retrieval Solution (Dako). Endogenous peroxidase was blocked using 3% hydrogen peroxide followed by an overnight incubation with primary mouse anti-HPD antibody (Santa Cruz sc-271672) diluted 1:100. Biotinylated secondary antibody was incubated for 30 min. Detection of HPD was performed using the M.O.M Vectastain kit and DAB detection kit (Vector Laboratories) according to the manufacturer's recommendations. Haematoxylin was used for counterstaining.
For immunofluorescence detection, endogenous biotin was blocked using Avidin/Biotin blocking kit (Vector laboratories) before incubation with biotinylated secondary antibody. Streptavidin-AlexaFluor488 (Thermo Fisher Scientific, cat# S32354) was incubated for 30 min at a dilution of 1:1,000. EdU was detected using the Click-iT Plus EdU Alexa Fluor 594 Imaging Kit (Molecular Probes) according to the manufacturer's instructions.
6
Macrophage Distribution Analysis in Murine Colons
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Immunohistochemistry (IHC) was performed for analysis of macrophage distribution in distal colons of mice. Formalin-fixed paraffin-embedded sections were de-paraffinized and rehydrated using xylene and ethanol-water gradient. Murine macrophage-specific F4/80 antibody staining was performed according to standard protocol. Briefly, tissues were treated with antigen retrieval using citrate buffer (microwave boiling in antigen unmasking solution), and the primary antibody F4/80 (purchased from ATCC) was applied overnight at 4 °C, followed by biotinylated secondary antibody for 1 h at room temperature. The ABC-HRP kit was used to amplify the signal and visualization of antibody was performed using the DAB detection kit (Vector Laboratories, Burlingame, CA, USA). Slides were counterstained with hematoxylin for 10 min and mounted with glass coverslips. F4/80+ cells was quantified in representative 10 randomly selected 200× field per specimen. Representative F4/80+ cells of distal colon per mice were calculated as the median number of all number of F4/80+ cells in individual measurements made for each mouse. Representative images were taken using an optical microscope and rendered using Adobe Photoshop (Adobe, San Jose, CA, USA).
7
Histological Analysis of Aortic Valve Plaques
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Hearts were embedded in OCT (Optimal Cutting Temperature; Tissue-Tek, Zoeterwoulde, The Netherlands) and sections of 10 μm thickness were collected. For macrophage and T cell content, subvalvular plaques were stained with MOMA-2 antibody (BMA Biomedicals, Switzerland) and anti-CD3 (Dako A0452) respectively, using rabbit immunoglobulins (Dako, Solna, Sweden) as negative controls. DAB detection kit was used for color development (Vector Laboratories, CA) and the sections were counterstained in haematoxylin. For assessment of the collagen content of the plaques, subvalvular sections were stained with Van Gieson Solution Acid Fuchsin (Sigma-Aldrich). To assess the necrotic core, sections were stained with haematoxylin/eosin and the area was determined as the acellular area, lacking nuclei and cytoplasm, under the fibrous cap of lesions. All stainings were quantified with Image-Pro-Plus 4.5 software (Media Cybernetics).
8
Immunostaining of Inner Ear Tissues
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Inner ears from RbloxP/loxP;Fgfr3-iCre-ERT2 mice were perilymphatically fixed with 4% PFA at P7, and immersed in the fixative overnight. Specimens were decalcified for 6 h in 0.5 M EDTA, pH 8.0, followed by embedding in paraffin and cutting to 5-μm-thick sections. Epitopes were unmasked by microwave heating (800 W) in 10 mM citrate buffer, pH 6.0, for 10 min. Sections were stained overnight with the γH2AX and rabbit monoclonal Ki-67 (LabVision/Thermo Scientific, #RM-9106, clone SP6, 1:250) antibodies in PBS-TX. Detection was done using the Mouse-on-Mouse (γH2AX) and Vectastain Elite ABC (Ki-67) kits and the DAB Detection kit (Vector Laboratories). Sections were counterstained with 3% methyl green and mounted in Permount (Fisher Scientific).
9
Histological Examination of Kidney Tissue
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Hematoxylin–eosin, periodic acid–Schiff (PAS), and Masson’s trichrome stainings were performed by conventional histochemical methods described previously [25 (link)]. After the mice were euthanized, full-thickness biopsies were taken and fixed in 4% formaldehyde, and then embedded in paraffin. Tissue slices were subjected to hematoxylin–eosin staining. For PAS staining, the kidneys were harvested and fixed in 10% formalin; 5-μm thick sections were stained with PAS reagent. Immunohistochemistry was performed in paraffin sections and detected in cortical sections with a specific primary antibody and the ABC staining kit, and visualized with the DAB detection kit (both kits from Vector Laboratories Inc., Burlingame, CA, USA). The primary antibody used in the present study was RAS (Sigma; Maixin, Fuzhou, China). After being incubated with the secondary antibody (Proteintech Group, Chicago, IL, USA), 2-μm thick sections were developed with an SP immunohistochemical kit (Maixin, China) to produce a brown product and counterstained with hematoxylin. Histologic evaluation was performed using a Nikon Eclipse E600 microscopy system (Nikon Instruments Inc., Melville, NY, USA) without knowledge of the identity of the various groups. The slides were coded and examined by a pathologist blinded to the study protocol to identify histological alterations.
10
Quantitative IHC Analysis of ACSL4 Expression
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IHC staining was performed as described in our recent studies (Shen et al., 2022a (link); Yao et al., 2022 (link)). Briefly, following deparaffinization and antigen retrieval, FFPE slides were blocked in phosphate-buffered saline (PBS) containing 4% donkey serum and 0.2% Triton X-100 for 1 h at room temperature. Then FFPE slides were incubated with primary antibody to ACSL4 at a dilution of 1:250 (Abcam, MA) overnight at 4°C; the sections were washed with PBS three times and incubated with biotinylated secondary donkey anti-rabbit antibody (Vector Laboratories, CA) in PBS with 0.2% Triton X-100 at a dilution of 1:500 for 1 h. Sections were washed again, and the secondary antibodies were detected by use of a DAB detection kit (Vector Laboratories), followed by counterstaining in 50% hematoxylin for 2 min.
ACSL4 staining in IHC was quantified with use of ImageJ, an image processing program developed at National Institutes of Health (Schneider et al., 2012 (link)). The mean of percentage of staining area by ACSL4 protein in the chorionic villi tissues was acquired from three random fields. We set the overall scores from 0 to nine based on the percentage staining area of <0.1%, ≥0.1 and <1%, ≥1 and <2%, ≥2 and <3%, ≥3% and <4%, ≥4 and <5%, ≥5 and <6%, ≥6 and <7%, ≥7 and <8%, and ≥9%, respectively.
ACSL4 staining in IHC was quantified with use of ImageJ, an image processing program developed at National Institutes of Health (Schneider et al., 2012 (link)). The mean of percentage of staining area by ACSL4 protein in the chorionic villi tissues was acquired from three random fields. We set the overall scores from 0 to nine based on the percentage staining area of <0.1%, ≥0.1 and <1%, ≥1 and <2%, ≥2 and <3%, ≥3% and <4%, ≥4 and <5%, ≥5 and <6%, ≥6 and <7%, ≥7 and <8%, and ≥9%, respectively.
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