After 28 days of culture, cells within the scaffolds were fixed in formalin (10% phosphate buffer, Fisher Scientific) and characterized by IHC for the expression of OCN. Heat-mediated antigen retrieval was performed with sodium citrate buffer (pH 6.0) for 20 min at 95 °C. Endogenous peroxidase was blocked with 3% H2O2 in methanol for 10 min, and nonspecific binding was suppressed with 1% horse serum in PBS for 45 min. Following antigen retrieval and blocking, the samples were incubated with rabbit anti-OCN primary antibody (1:200) (Abcam ab93876, Cambridge, MA) or an isotype control antibody (Abcam) overnight at 4 °C, followed by 30 min of incubation with horse anti-rabbit biotinylated secondary antibodies (Vector Laboratories, Burlingame, CA). Staining was developed by incubating the samples with horseradish peroxidase-conjugated streptavidin (Vector Laboratories) and treating the samples with the Vector® NovaRED™ peroxidase substrate (Vector Laboratories). Following IHC, the samples were counterstained with hematoxylin, dehydrated, mounted, and coverslipped, and images were captured with an Olympus CKX41 microscope (Center Valley, PA).
Vector novared peroxidase substrate
Manufactured by Vector Laboratories
Sourced in Germany, United Kingdom
Vector NovaRED peroxidase substrate is a chromogenic substrate used for the detection of peroxidase-conjugated antibodies or other biomolecules in immunohistochemistry, enzyme-linked immunosorbent assays (ELISAs), and other applications. It produces a red-colored precipitate at the site of the peroxidase enzyme reaction.
Automatically generated - may contain errors
Lab products found in correlation
Ckx41 microscope, by Olympus (3 mentions)
Horseradish peroxidase conjugated streptavidin, by Vector Laboratories (3 mentions)
Rabbit anti ocn primary antibody, by Abcam (2 mentions)
Horse anti rabbit biotinylated secondary antibodies, by Vector Laboratories (2 mentions)
Horse serum, by Vector Laboratories (2 mentions)
Elispot plate, by Merck Group (2 mentions)
Il 17a, by Thermo Fisher Scientific (2 mentions)
Elispot diluent, by Thermo Fisher Scientific (2 mentions)
Ifn γ, by BD (2 mentions)
Pepmix sars cov 2, by JPT Peptide Technologies (2 mentions)
Isotype control antibody, by Abcam (1 mentions)
Formalin, by Thermo Fisher Scientific (1 mentions)
Citrate buffer ph 6, by Merck Group (1 mentions)
Biotin conjugated secondary antibody, by Vector Laboratories (1 mentions)
Shandon harris hematoxylin, by Thermo Fisher Scientific (1 mentions)
Vectastain elite abc kit, by Vector Laboratories (1 mentions)
Ab56416, by Santa Cruz Biotechnology (1 mentions)
Igg2a isotype control, by Santa Cruz Biotechnology (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Alexa fluor 488 conjugated secondary antibody, by Abcam (1 mentions)
13 protocols using vector novared peroxidase substrate
1
Immunohistochemical Analysis of Osteocalcin Expression
2
Cranial Bone Defect Histological Analysis
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The rat samples of cranial bone defects were collected and fixed with 4% paraformaldehyde solution for 24 h. Calvarium were decalcified using 14% ethylene diamine tetraacetic acid at pH 7.4 for 4 days. Then, the cranial bone was embedded in paraffin solution and cut longitudinally into a thickness of 5 μM. After the sections were made, performed them with H&E staining and Masson staining. Samples were also obtained for immunohistochemical (IHC) staining to analyze the expression of the vascular endothelial growth factor (VEGF), collagen type-1 (COL-1), and osteocalcin (OCN). Briefly, sodium citrate buffer was used to retrieve antigen for 20 min at 95°C, thus suppressing nonspecific binding for 1 h at room temperature. Samples were incubated with corresponding primary antibody overnight at 4°C and then incubated samples and secondary antibodies for 30 min. Samples were incubated with horseradish peroxidase-conjugated streptavidin (Vector Laboratories) and treated with the Vector NovaRED peroxidase substrate (Vector Laboratories). After IHC, samples were then subsequently counterstained with hematoxylin, dehydrated, mounted, and cover slipped. The images were captured by the Olympus CKX41 microscope.
3
Immunohistochemical Analysis of Buruli Ulcer Biopsies
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Fixed and embedded 4 mm punch biopsies from Buruli ulcer patients were described previously [6 (link)]. In ulcerated BU lesions (6 patients) punch biopsies were taken 1 cm inside the outer margin of the induration surrounding the ulcer. For BU plaque lesions (2 patients) punch biopsies were collected from the non-ulcerated center of the lesion. Healthy control tissue was obtained at The Whitely Clinic or purchased from AMS Biotechnology (Europe) Ltd (500041028). After removal, all tissues were fixed in neutral formalin, embedded into paraffin (pfm Medical, 9000R2010) and sectioned (5 μm). Sections were deparaffinized, endogenous peroxidase quenched, epitopes unmasked (pH 6 citrate buffer; Sigma, C999) and blocked with horse serum (Vector Laboratories, S-2000-20). The tissue sections were then incubated with either anti-SQSTM1 antibody (Abcam, ab56416) or IgG2a isotype control (Santa Cruz Biotechnology, sc-3878) overnight at 4°C and biotin-conjugated secondary antibody (Vector Laboratories, BA2000). Staining was performed using VECTASTAIN Elite ABC kit (Vector Laboratories, PK-6100) and Vector NovaRED peroxidase substrate (Vector Laboratories, SK-4800). Counterstaining was performed with Shandon Harris Hematoxylin (Thermo Fisher Scientific, 6765001).
4
Immunohistochemistry and Immunofluorescence Protocols
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For immunohistochemistry (IHC), rehydrated slices were processed for antigen retrieval by heating in diluted 1× antigen retrieval solution (eBioscience, San Diego, CA) at 90°C for 20 min. The samples were blocked with 10% horse serum (Vector Labs, Burlingame, CA, USA), then incubated with primary antibodies (Table S2 ) at 4°C overnight. On the next day, slices were washed with PBS and incubated in biotinylated secondary antibody for an hour. Then the slices were incubated with horseradish peroxidase‐conjugated streptavidin for another 0.5 h and visualized by Vector NovaRED™ peroxidase substrate. Hematoxylin was used for counterstaining (Vector Labs). Images were acquired with a Nikon Eclipse E800 upright microscope (Nikon, Melville, NY).
For immunofluorescence staining (IF), samples were first penetrated by 0.02% Triton X‐100 (Sigma‐Aldrich) for 10 min. After being blocked with 5% BSA, slides were exposed to primary antibodies (TableS2 ) overnight at 4°C. Alexa Fluor® 488‐conjugated Secondary antibody was used (Abcam, Branford, CT, United States). 4′,6‐diamidino‐2‐phenylindole (DAPI)‐containing antifade medium (Vector Labs) was utilized to mount the slides. An EVOS M5000 microscope (Thermo Fisher Scientific) was used to image the stained sections.
For immunofluorescence staining (IF), samples were first penetrated by 0.02% Triton X‐100 (Sigma‐Aldrich) for 10 min. After being blocked with 5% BSA, slides were exposed to primary antibodies (Table
5
Immunohistochemical Analysis of TLR9 Expression
6
Histological Analysis of Cranial Defect Repair
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At 6 weeks after cranial defect treatment, the samples were collected for hematoxylin and eosin (H&E) staining. The samples were fixed by 10% formalin for 24 hours. After that, 10% EDTA‐2Na+ 1% NaOH were utilized to decalcify for 24 hours, and then were embedded in paraffin and sectioned (8 μm thicknesses). Samples were also collected for immunohistochemical (IHC) stainings to analyses the expression of the OCN gene. Briefly, sodium citrate buffer was used to retrieve antigen for 20 minutes at 95°C, nonspecific binding was suppressed with for 1 hour at room temperature. Samples were incubated with rabbit anti‐OCN primary antibody (1:200) (Abcam ab93876, Cambridge, Massachusetts) overnight at 4°C, then incubated with horse anti‐rabbit biotinylated secondary antibodies (Vector Laboratories, Burlingame, California) 30 minutes. Samples were incubated with horseradish peroxidase‐conjugated streptavidin (Vector Laboratories) and treated with the Vector NovaRED peroxidase substrate (Vector Laboratories). After IHC, samples were then subsequently counterstained with hematoxylin, dehydrated, mounted, cover slipped. The images were captured by the Olympus CKX41 microscope.
7
Immunohistochemistry with HRP-Conjugate Kits
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Three commercial, horseradish peroxidase (HRP)-linked conjugate detection kits were utilized by following manufacturer’s instructions. These kits included the Vectastain® ABC Kit–Mouse IgG (Vector Laboratories, Burlingame, CA, USA), the Vectastain® ABC Kit–Rabbit IgG, and the Novolink™ Polymer Detection System (Leica, Wetzlar, Germany).
Lastly, a substrate-chromogen (Vector NovaRED Peroxidase Substrate, Vector Laboratories) was applied for 10 min, rinsed with de-ionized water for 5 min, counterstained with Lab Vision™ Mayer’s Hematoxylin (ThermoFisher Scientific) for 1 min, and rinsed in running tap water for 2 min. All sections were then dehydrated through an increasing gradient of ethanol, 50%, 70%, 95%, and 100%, for 2 min each. Slides were cleared in xylene three times for 3 min each before coverslipping with Permount™ mounting medium (Fischer Scientific, Hampton, NH, USA).
Lastly, a substrate-chromogen (Vector NovaRED Peroxidase Substrate, Vector Laboratories) was applied for 10 min, rinsed with de-ionized water for 5 min, counterstained with Lab Vision™ Mayer’s Hematoxylin (ThermoFisher Scientific) for 1 min, and rinsed in running tap water for 2 min. All sections were then dehydrated through an increasing gradient of ethanol, 50%, 70%, 95%, and 100%, for 2 min each. Slides were cleared in xylene three times for 3 min each before coverslipping with Permount™ mounting medium (Fischer Scientific, Hampton, NH, USA).
8
Immunohistochemical Analysis of Mouse IgG
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Thirty-five μm-thick sections were washed in 0.01 M PBS and then incubated in 3% hydrogen peroxide (methanol: 30% H2O2 = 1:1 in 0.01 M PBS) for 20 min and permeabilized with PBS containing 0.25% Triton X-100 (PBST, 3 × 10 min). The sections were blocked in 10% normal goat serum at room temperature (RT) for 1 h, followed by incubation with biotinylated-goat anti-mouse IgG (#BA9200, Vector Laboratories, Burlingame, CA, USA) overnight at 4°C. Free-floating sections were subsequently washed in permeabilizing buffer three times. For detection of mouse IgG, sections were incubated in avidin Vectastain ABC KIT (#sk4600, Vector Laboratories) for 30 min and revealed for 5 min using Vector NovaRed peroxidase substrate (#sk4800, Vector Laboratories). The reaction was stopped with PBS (3 × 5 min), and sections were mounted to a glass slide. Then samples were air dried in air overnight and dehydrated using isopropanol xylene before being cover slipped with Aqua/polymount (Polyscience, Philadelphia, PA, USA). Images were captured under a light microscope using a 2X lens (B-Z Series, Keyence, Osaka, Japan). Pixel intensity was calculated using Fiji software.
9
Immunohistochemical Analysis of NLRP3 and Caspase-1
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Staining for NLRP3 and cleaved caspase-1 was performed using immunohistochemistry. After antigen retrieval, endogenous peroxidases were inactivated by a solution of 3% hydrogen peroxide for 15 min. A primary antibody raised against mature, active caspase-1 (cleaved Asp210, 1:50; Thermo Fisher Scientific, Waltham, Massachusetts) or NLRP3 (1:100; Novus Biologicals, Centennial, Colorado) was incubated overnight at 4°C. An anti-rabbit IgG conjugated to the horseradish peroxidase secondary antibody was incubated for 2 h at room temperature, followed by incubation with VECTOR NovaRED Peroxidase Substrate (1:100; Vector Laboratories, Burlingame, California) and counterstaining with hematoxylin. Finally, slides were dehydrated and coverslipped with Cytoseal XYL (Thermo Fisher Scientific). Images were acquired using a Zeiss Axio Imager A.1 microscope (Zeiss, Oberkochen, Germany) using 10× objective for immunohistochemistry. Quantification was performed as previously mentioned (Supplemental Table 1 ).
10
Hearing Evaluation and Cochlear Histology
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Two weeks after injection of the drug into the middle ear cavity, the animals' hearing was evaluated by using the auditory brainstem response (ABR); afterward, the cochlea and bulla were collected, and histological damage was observed with an optical microscope. Cochleae and bullae were fixed in 4% paraformaldehyde (Merck, Darmstadt, Germany) and decalcified in 5% ethylenediaminetetraacetic acid (EDTA, 0.3 M). Cochlea sections on slides were stained with hematoxylin (YD Diagnostics Corp., Yongin-si, Republic of Korea) and eosin (BBC Biochemical, McKinney, TX). Images were examined using a light microscope system (Nikon Eclipse E400, Tokyo, Japan).
Immunofluorescence staining for dexamethasone was performed to examine the absorption of dexamethasone in the cochlear tissue (Yang et al., 2018 (link)). Anti-dexamethasone antibody (Abcam, Cambridge, UK) was used as the primary antibody, and the tissue was stained using the Vectastain Elite ABC HRP Kit and the Vector NovaRED Peroxidase Substrate (Vector Laboratories, Burlingame, CA). Images were captured using a light microscope system (Nikon Eclipse E400, Tokyo, Japan).
Immunofluorescence staining for dexamethasone was performed to examine the absorption of dexamethasone in the cochlear tissue (Yang et al., 2018 (link)). Anti-dexamethasone antibody (Abcam, Cambridge, UK) was used as the primary antibody, and the tissue was stained using the Vectastain Elite ABC HRP Kit and the Vector NovaRED Peroxidase Substrate (Vector Laboratories, Burlingame, CA). Images were captured using a light microscope system (Nikon Eclipse E400, Tokyo, Japan).
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