Free-floating sections were selected based on previous FISH labeling to provide verification of common protein marker identification. Sections selected for KChIP1 were rinsed in Phosphate Tris (PT, pH 7.2-7.4) buffer and Phosphate-Buffered Saline with 0.2% Triton (PBST, pH 7.2-7.4) respectively. They were then moved into a 0.5% H2O2 buffer for 10 minutes followed by a series of rinses before incubating in a 10% Normal Horse Serum (NHS, Vector, S-2000) solution for one hour. The tissue was transferred to primary antibody solution (NeuroMab, Anti-KChIP1, clone K55/7, 1:200) for overnight incubation at 4°C. The next day, sections were again serially rinsed in respective buffers and incubated in secondary antibody (Vector, Vectastain ABC, Peroxidase Kit, PK-4002, 1:200) for 30 minutes. Sections were again serially rinsed and placed into ABC (Vector, Vectastain ABC, Peroxidase Kit, PK-4002) buffer for one hour incubation before being rinsed. Tissue was placed in DAB substrate solution (3,3’-diaminobenzidine, Vector, SK-4100) until reacted. Following a final serial rinse, sections were mounted for imaging and analysis.
Vectastain abc peroxidase kit
Manufactured by Vector Laboratories
Sourced in United States
The Vectastain ABC peroxidase kit is a labeling system used for the immunohistochemical detection of antigens in fixed tissue sections. The kit contains the necessary components to perform the avidin-biotin-peroxidase complex (ABC) method, which is a widely used technique for signal amplification in immunohistochemistry.
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Lab products found in correlation
F4 80, by Bio-Rad (2 mentions)
3 3 diaminobenzidine substrate, by Merck Group (2 mentions)
Keratin 6, by Fortrea (2 mentions)
Lactoferrin, by Abcam (2 mentions)
Hmgb1, by R&D Systems (2 mentions)
Hsp70, by BioGenex (2 mentions)
Dab peroxidase substrate kit, by Vector Laboratories (2 mentions)
Zen blue software, by Zeiss (2 mentions)
Anti timp 1, by R&D Systems (1 mentions)
Anti mmp12, by Abcam (1 mentions)
Anti mmp 13, by R&D Systems (1 mentions)
Vector hematoxylin qs, by Vector Laboratories (1 mentions)
Anti timp2, by Abcam (1 mentions)
Dab substrate kit, by Vector Laboratories (1 mentions)
Tissue freezing medium, by Leica (1 mentions)
Diaminobenzidine peroxidase substrate kit, by Vector Laboratories (1 mentions)
Axio imager a1 microscope, by Zeiss (1 mentions)
Vt1200 s vibrating blade microtome, by Leica (1 mentions)
Dab staining kit, by Merck Group (1 mentions)
Vector dab, by Vector Laboratories (1 mentions)
25 protocols using vectastain abc peroxidase kit
1
Immunohistochemistry for KChIP1 Protein
2
Immunohistochemical Analysis of Liver ACHE
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After fixation of liver and spleen specimens in 10% buffered neutral formalin for 72 h, the specimens were washed, dehydrated in graded series of alcohol and cleared in xylol, and then embedded in paraffin. Serial sections of 4-5-µm thickness were obtained and stained with hematoxylin and eosin [19 ]. Electric light microscope Olympus BH2 (Tokyo, Japan) was used for the histopathological examination of hematoxylin and eosin sections.
Expression of ACHE in paraffin sections of hepatic tissue of all groups was demonstrated immunohistochemically according to the method described by Hsu et al. [20 ] using avidin-biotin-peroxidase (3,3-diaminobenzidine tetrahydrochloride [DAB], Sigma Chemical Co.).
After incubation of liver sections with monoclonal antibody for ACHE (Dako Corp, Carpinteria, CA) and reagents of avidin-biotin-peroxidase method (Vectastain ABC Peroxidase Kit, Vector Laboratories), antigen-antibody complex was detected. The immunoreactive cells were visualized using chromagen DAB (Sigma Chemical Co.). Following examination, the intensity of immunohistochemical staining of ACHE was quantified in random five high microscopic fields as an optical density using image analysis software (Image J, 1.46a, NIH, USA).
Expression of ACHE in paraffin sections of hepatic tissue of all groups was demonstrated immunohistochemically according to the method described by Hsu et al. [20 ] using avidin-biotin-peroxidase (3,3-diaminobenzidine tetrahydrochloride [DAB], Sigma Chemical Co.).
After incubation of liver sections with monoclonal antibody for ACHE (Dako Corp, Carpinteria, CA) and reagents of avidin-biotin-peroxidase method (Vectastain ABC Peroxidase Kit, Vector Laboratories), antigen-antibody complex was detected. The immunoreactive cells were visualized using chromagen DAB (Sigma Chemical Co.). Following examination, the intensity of immunohistochemical staining of ACHE was quantified in random five high microscopic fields as an optical density using image analysis software (Image J, 1.46a, NIH, USA).
3
Immunohistochemical Analysis of MMPs and TIMPs
4
Immunohistochemical Analysis of Langerin Expression
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Wounds were excised, bisected, and directly frozen in tissue freezing medium (Leica Biosystems, Wetzlar, Germany) without prior fixation. Frozen sections (7 μm) were fixed for 10 min with 4% paraformaldehyde and analyzed by immunohistochemistry staining [39 ] using rat‐anti‐langerin/CD207 IgG2a (eBioL31, Thermo Fisher), the Vectastain ABC peroxidase kit, and the diaminobenzidine peroxidase substrate kit (both from Vector Laboratories, Burlingame, CA).
5
Histological Analysis of Prion Proteins
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Brains were removed, immersion-fixed in 10% buffered formalin, and then embedded in paraffin. Sections were cut at 8 µm, mounted on glass slides, deparaffinized, and then processed for immunohistochemistry or stained with hematoxylin and eosin (H&E). Endogenous tissue peroxidases were inhibited by incubating the slides in a 3% hydrogen peroxide solution (prepared in methanol) for 30 min. Sections to be stained with anti-PrP antibodies were subjected to hydrolytic autoclaving (121°C for 10 min in citrate buffer). Slides were then blocked with 10% (vol/vol) normal goat serum for 1 h and then incubated with primary antibody overnight at 4°C. The following primary antibodies were used: anti-GFAP rabbit polyclonal antibody Z0334 (Dako, 1∶500 dilution) to detect astrocytic gliosis, and anti-PrP antibodies 3F4 (1∶1,000 dilution) [83] (link) or HuM-D18 (1∶500 dilution) [84] (link) to detect PrPSc deposition. Bound antibody was detected using a Vectastain ABC peroxidase kit (Vector Laboratories) and visualized using 3-3′-diaminobenzidine (DAB). Slides were counterstained with hematoxylin and then photographed using an AxioImager.A1 microscope (Carl Zeiss).
6
Histopathological Assessment of Skin and Tumor Tissues
7
Immunohistochemical Analysis of GFP Expression
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After postfixation in 4% paraformaldehyde (PFA), brains were sectioned into 50-μm sections using a Leica VT1200S vibrating-blade microtome. Brains were then blocked and permeabilized in 10% normal goat serum and 1% Triton X-100 in 1× PBS for 1 h at room temperature. Primary antibody staining was performed with mouse anti-GFP antibody overnight at 4°C. Brains were then stained with 3,3′-diaminobenzidine (DAB) using a Vectastain ABC peroxidase kit (PK-4001) from Vector Laboratories (Burlingame, CA) according to the manufacturer’s instructions. Development of the stained tissues was carried out using the DAB staining kit (D7304) from Sigma-Aldrich (St. Louis, MO). Slides were imaged by the UNC at Chapel Hill Translational Pathology Laboratory using the Aperio ScanScope XT system. Images were analyzed using the Aperio ImageScope software, and quantification was performed using ImageJ software.
8
Immunohistochemical Analysis of Kisspeptin, Androgen Receptor, and Inflammatory Markers
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Paraffin-prepared sections were stained using the avidin–biotin peroxidase system for the Kisspeptin, androgen receptor, TNFα, Nrf2, and Ki67 detection using a primary antibody (rabbit polyclonal antibody). Following three PBS washes (5 min each), the sections were incubated for 30 min at room temperature with biotinylated secondary antibody and avidin–biotin complex (Vectastain® ABC-peroxidase kit, Vector Laboratories, Burlingame, CA). The color was developed using 3,3′ -diaminobenzidine (DAB) substrate (Vector® DAB, Vector Laboratories). Mayer’s hematoxylin was used as a counterstain. The stained sections were examined by light microscopy (LEICA ICC50 W) in the Image Analysis Unit of the Human Anatomy and Embryology Department, Zagazig University (Khamis et al., 2023 (link)).
9
Tyrosine Hydroxylase Immunohistochemistry
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Immunohistochemistry was performed as described previously (Seiler et al., 2016 (link)). The brains were cut at 30 μm on a cryostat (Leica CM 1900) and mounted on Superfrost slides (Thermo Scientific). Sections were heated in citrate buffer for 30 min and blocked with 10% horse serum in 0.1% Triton-PBS. Primary and secondary antibodies were incubated in a 0.1% Triton-PBS solution containing 2.5% horse serum. Slides were incubated with the mouse monoclonal anti-tyrosine hydroxylase (TH; 1:1000, Millipore) overnight. After PBS washes, sections were incubated for 2 h with the secondary biotinylated anti-mouse IgG antibody (1:200, Vector Laboratories) and the endogenous peroxidase blocked with a solution of 10% methanol and 3% hydrogen peroxide in PBS. Thereafter, the slides were incubated with an avidin-biotin-complex (7 μl/ml; Vectastain ABC-Peroxidase KIT, Vector Labs) for 1 h and specifically bound antibodies were visualized with a metal-enhanced 3,3′-diaminobenzidine substrate kit (Pierce, 34002, Life Technologies). The sections were dehydrated in alcohol, cleared in xylene and mounted in Eukitt (O. Kindler GmbH, Freiburg, Germany).
10
Immunohistochemistry of HMGB1, RAGE, and HSP70
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Sections from formalin fixed tissues in paraffin blocks were deparaffinised and rehydrated before microwave-assisted antigen retrieval in citric acid buffer at pH 6.0. Endogenous peroxidase activity was blocked with CH3OH/H2O2 treatment and were blocked with 2% BSA in PBS. Sections were incubated overnight at 4°C with the primary antibody diluted in 2% serum in PBS(7 (link)). We used the following primary antibodies: Lactoferrin (Abcam, Cambridge, MA, dilution 1:50), HMGB1 (R&D Systems, Minneapolis, MN, dilution 1:50), RAGE (AbD Serotec, Oxford, UK, dilution 1:1000), HSP70 (Biogenex, San Ramon, CA, dilution 1:50). Sections were then washed and incubated with the appropriate species-specific secondary antibody diluted 1:200 in 2% serum for 2 hours at room temperature. After further washing, antigen:antibody complexes were visualized using a Vectastain ABC peroxidase kit (Vector Laboratories Inc., Burlingame, CA). Antigen detection was enhanced with nickel-diaminobenzidine, followed by incubation with TRIS-cobalt. Slides were counterstained with Nuclear Fast Red for photomicroscopy.
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