For immunohistochemistry analysis, we collected tissue samples (the brain, lung, liver, spleen, kidney, intestine and spinal cord) at 6 dpi from CB1/2014-infected young adult or aged ferrets. The samples were fixed in 10% neutral-buffered formalin and embedded in paraffin according to the standard procedures. The embedded tissues were sectioned and dried for 3 days at room temperature. To detect the viral antigen by immunohistochemistry, a monoclonal antibody against SFTSV was used as the primary antibody. Antigen was visualized using the biotin-avidin system (Vector Labs). Slides were viewed using the Olympus IX 71 (Olympus) microscope with DP controller software to capture images.
Avidin biotin system
Manufactured by Vector Laboratories
Sourced in United States
The Avidin-biotin system is a powerful and versatile tool used in various biochemical and molecular biology applications. It is based on the strong non-covalent interaction between the protein avidin and the small molecule biotin. This interaction is one of the strongest known between a protein and a ligand, with a dissociation constant in the femtomolar range. The Avidin-biotin system can be used for the detection, purification, and immobilization of biomolecules, such as proteins, nucleic acids, and carbohydrates.
Automatically generated - may contain errors
Lab products found in correlation
Avidin biotin blocking kit, by Vector Laboratories (1 mentions)
3 3 diaminobenzidine (dab), by Vector Laboratories (1 mentions)
Biotinylated horse anti mouse secondary antibody, by Vector Laboratories (1 mentions)
Aquatex, by Merck Group (1 mentions)
Biotinylated secondary antibody, by Vector Laboratories (1 mentions)
Perk antibody, by Cell Signaling Technology (1 mentions)
Immpact novared hrp substrate, by Vector Laboratories (1 mentions)
Vectastain abc kit, by Vector Laboratories (1 mentions)
8 protocols using avidin biotin system
1
Immunohistochemical Analysis of SFTSV in Ferrets
2
SARS-CoV-2 Antigen Detection in Tissues
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Tissue samples were collected from PBS control and SARS-CoV-2-infected ferrets or mice as previously described (52 (link)). Tissues were incubated in 10% neutral buffered formalin for fixation before they were embedded in paraffin based on standard procedures as described before. The embedded tissues were sectioned and dried for 3 days at room temperature. To detect the viral antigen by immunohistochemistry (IHC), rabbit polyclonal antibody of ORF8 purchased from GeneTex was used as the primary antibody. Antigen was visualized using the biotin-avidin system (Vector Labs). Slides were viewed using the Olympus IX 71 (Olympus, Tokyo, Japan) microscope with DP controller software to capture images (×400).
3
Immunohistochemical Localization of Taurine in Fetal Mouse Neocortex
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Taurine localization was observed immunohistochemically. The fetal neocortex of E17.5 mice was fixed via perfusion with 4% paraformaldehyde/0.5% glutaraldehyde in 0.1 M Tris buffer (TB). The brains were removed and immersed in the same fixative for 3 h at 4°C. After post-fixing, the brains were transferred to 15% sucrose TB for 1 h and 30% sucrose TB for 3 days. Tissues were cut coronally with a cryostat into sections with a thickness of 30 μm and rinsed in Tris-buffered saline (TBS). The sections were first treated with 0.5% NaBH4, then incubated for 0.5 h in 0.3% H2O2 in TBS and subsequently with an antibody against taurine (TT100, polyclonal rabbit IgG; Signature Immunologics, Salt Lake City, UT, USA) for 36 h at 4°C. After rinsing in TBS, the sections were incubated for 1 h with a secondary antibody against rabbit IgG (anti-rabbit biotinylated, BA1000; Vector Laboratories, Burlingame, CA, USA). The primary and secondary antibodies were diluted in TBS containing 2% horse serum albumin and 0.1% Triton X-100. Visualization was performed using the Biotin/Avidin system (Vector Laboratories). Sections were treated in diaminobenzidine (DAB). After rinsing, the sections were mounted on slides and coverslipped and finally observed under a microscope (AX-80; Olympus) equipped with a CCD camera (EP-70; Olympus).
4
Immunohistochemical Analysis of VEGFR-2 and PCNA
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In some experiments, formalin-fixed, paraffin-embedded RV sections were stained for VEGFR-2 expression or proliferating cell nuclear antigen (PCNA) following antigen retrieval with citrate-EDTA buffer [28 (link)]. Non-specific binding was blocked by incubation with 5% normal goat serum, and endogenous biotin, biotin receptors, and avidin binding sites were blocked using the Avidin/Biotin Blocking Kit (Vector Laboratories; Burlingame, CA). Primary antibodies (Cell Signaling Technology cat. #9698, 1:200; #13110, 1:500) were diluted in TBST containing 1% normal goat serum and applied to sections for 40 min at room temperature. Primary antibody was detected using an avidin-biotin system (Vector Laboratories;), and HRP activity was visualized with 3,3′-diaminobenzidine or NovaRed substrates (Vector Laboratories).
5
Immunohistochemical Quantification of Ki-67
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From each formalin-fixed and paraffin-embedded sample, 5 μm-thick sections were cut. Histological sections were deparaffinized and rehydrated using graded alcohols, and endogenous peroxidase activity was blocked for 30 min in a 0.3% H2O2 methanol solution. Heat-induced antigen retrieval was performed using a pressure cooker for 20 min in a citrate buffer (pH 6.0). After washing in Tris-buffer, protein blocking was performed using normal horse serum for 30 min at 25°C. Sections were then incubated with a mouse anti-human Ki-67 primary antibody (MIB-1; Dako, CA, USA) diluted 1:600 in Tris buffer for 18 h at 4°C. After rinsing sections in Tris buffer, slides were incubated with a biotinylated horse anti-mouse secondary antibody (Vector Laboratories, CA, USA) for 30 min at 25°C. Immunohistochemical signals were detected using an avidin-biotin system (Vector Laboratories, CA, USA) and 3-amino-9-ethylcarbazole (AEC) substrate-chromogen kit (Vector Laboratories, CA, USA). Sections were counterstained with Harris hematoxylin and mounted using an aqueous mounting medium (Aquatex, Sigma-Aldrich, MO, USA). The Ki-67 value was expressed as the percentage of positively stained cells, calculated by counting 1,000 cells per section (400× magnification).
6
Immunohistochemical Analysis of iNOS Expression
7
Immunohistochemical Analysis of Neural Signaling
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Mice were anesthetized by intraperitoneal injection of urethane (1.6 g/kg bodyweight), transcardially perfused with 4% paraformaldehyde in phosphate buffered saline (PBS, pH 7.4) and brains were dissected and postfixed overnight in 4% paraformaldehyd. 40μm thick sections were cut using a vibratom (Leica) and incubated with pERK antibody (Cell Signalling #9101), dilution 1:1000; Arc/Arg3.1 antibody58 (link), dilution 1:1500 or p-CaMKII (Thr286)(D21E4) (Cell Signalling #12716), dilution 1:3000. Immunoreactivity was detected using the avidin-biotin system (Vectastain, Vector Labs). Finally, sections were developed using diaminobenzidin (Sigma) as a chromogen. All experiments were performed with four replicate animals.
8
Immunohistochemical Analysis of AID and PRMT1
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Sections of 5-μm of paraffin-embedded tissues were deparaffinized in two changes of xylene for 5 min each and then rehydrated in distilled water using graded alcohols. Antigen retrieval was done by steaming the slides for 20 min then cooling for 20 min in EDTA buffer (1 mM EDTA, 0.05% Tween 20, pH 8) for AID and PRMT1. Endogenous peroxidase was blocked with a 0.3% hydrogen peroxide solution for 10 min. Endogenous biotin was blocked for 15 min with the blocking buffer provided with the Avidin/Biotin System (#SP2001; Vector Laboratories). For protein block, we used 10% normal goat serum and 1% BSA for 60 min at room temperature. Sections were incubated with anti-AID (1:50, rat Mab mAID-2 eBioscience), anti-PRMT1 (1:100) overnight at 4°C. Biotin-conjugated secondary antibodies were mouse anti–rabbit IgG (1:200; Vector Laboratories) to detect anti-PRMT1; mouse anti–rat IgG (1:200; Vector Laboratories) to detect anti-AID. Biotinylated reagents were detected with Vectastain ABC kit (PK-6100; Vector Laboratories). Peroxidase activity was developed using ImmPACT NovaRED HRP substrate (Vector Laboratories). Sections were counterstained with hematoxylin (cat. #MHS32-1L; Sigma-Aldrich) for 1 min prior to dehydrating and mounting for imaging on a bright field microscope.
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