The lung was inflated with 0.5% low-melting agarose and fixed using 4% formalin, and then embedded in paraffin and cut into 4-μm-thick sections for immunohistochemistry. The sections were stained using an Immunohistochemistry accessory kit (Bethyl Laboratories, Inc., USA) with anti-Egr-1 antibody (1:250, Abcam, UK) and then observed by microscopy.
Immunohistochemistry accessory kit
Manufactured by Fortis Life Sciences
Sourced in United States
The Immunohistochemistry accessory kit is a set of laboratory equipment designed to support immunohistochemistry techniques. The kit provides necessary components for sample preparation, staining, and visualization. It is intended for use in research and diagnostic applications involving the identification and localization of specific proteins or antigens within tissue sections.
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5 protocols using immunohistochemistry accessory kit
1
Lung Immunohistochemistry for Egr-1
2
Immunohistochemical Analysis of Inflammatory Markers
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IHC staining was performed using the immunohistochemistry accessory kit (Bethyl Laboratories, Montgomery, TX, USA). Each primary antibody was used at a 1:200 dilution according to the protocol. Antibody HIF-α was purchased from Novus (Novus Biologicals, Centennial, CO, USA). The antibodies to TNF-α, IL-6, RAGE, OPG, BMP-2, and BMP-7 were purchased from Bioworld (Bioworld Technology, Louis Park, MN, USA). RANKL antibody was purchased from Enzo (Enzo Life Sciences, Farmingdale, NY, USA). The levels of antibody expression were measured with software Image J (National Institutes of Health).
3
Tissue Immunohistochemistry for FIH-1 Expression
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Tissue sections were prepared by Drs. C. David James and Jann Sarkaria's group at Mayo Clinic [46] (link), [50] . An immunohistochemistry accessory kit (Bethyl laboratories, Inc., Montgomery, TX) was used to examine expression levels of FIH-1 according to the manufacturer's protocol. Briefly, after deparaffinization and rehydration, the slides were incubated in 3% hydrogen peroxide/methanol to quench endogenous peroxidase. Slides were then incubated in hot Epitope retrieval buffer (provided in kit) to recover epitopes for 20 min at 90–96°C. Non-specific reactions were blocked by incubating the sections with blocking reagent (included in the kit) for 30 min at room temperature, then 200 µl of human FIH-1 antibody (dilution 1∶200,catalog ab92498, Abcam, Cambridge, MA) was applied to each slide and incubated overnight at 4°C. Slides were then incubated with working anti-rabbit IHC antibody (provided in kit), and peroxidase activity was visualized with working 3, 3′-diaminobenzidine (DAB) substrate (provided in kit). Counterstaining was performed with hematoxylin. The stained sections were randomly selected high-power fields (×40).
4
Histological Analysis of Mandibular Tissue
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The mandibles were isolated and fixed in 10% neutral-buffered formalin solution. After fixation, the tissues were decalcified in 15% EDTA and 0.1 M Tris (pH 7.0). After decalcification, the implant was removed. The tissues were dehydrated with different percentage of alcohol, cleared in xylene, and embedded in paraffin. Tissue sections of 8 μm were mounted on glass slides and stained with hematoxylin and eosin (H&E) and immunohistochemical (IHC) stains. IHC staining was performed to detect the expression of IGFBP-3, BMP-2, BMP-7, osteoprotegerin (OPG), receptor activator of nuclear factor-κB ligand (RANKL), and receptor for advanced glycation end products (RAGE) using an immunohistochemistry accessory kit (Bethyl Laboratories, Montgomery, TX, USA). The primary antibodies were used at 1:200 dilutions according to the protocol. The slides were visualized microscopically (Carl Zeiss, Ostalbkreis, Germany). The levels of antibody expression were measured with ImageJ (National Institutes of Health, Bethesda, MD, USA) software.
5
Immunohistochemical Analysis of Bone Implants
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The mandibles with the implants were isolated and fixed in 10% neutral-buffered formalin solution. After decalcification in 15% ethylenediaminetetraacetic acid and 0.1 M Tris (pH 7.0), the implants were gently removed, and the mandibular tissues were dehydrated, cleared, and embedded in paraffin. Tissue sections, 8 mm in thickness, were mounted on glass slides and subjected to immunohistochemical (IHC) and hematoxylin and eosin (H&E) staining. IHC staining was performed to detect the expression of bone morphogenesis protein (BMP)-2, BMP-7, and collagen, using the immunohistochemistry accessory kit (Bethyl Laboratories, Montgomery, TX, USA). Collagen analysis was performed via Masson's trichrome staining using a commercial kit (Masson's trichrome stain kit, Sigma-Aldrich). The primary antibodies of BMP-2 (BMP2 P275 pAb, Bioworld Technology, Inc., USA) and BMP-7 (BMP7 E173 pAb, Bioworld Technology) were diluted in the ratio 1:100 for 30 min at room temperature in accordance with the manufacturer's protocol. The stained specimens were visualized microscopically (Carl Zeiss, Oberkochen, Germany) and cell counts in the newly formed bone area were obtained, in a 0.5 mm area surrounding the implants.
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