Tissue sections were deparaffinized and rehydrated through xylene and graded ethanols. Slides went through heat antigen retrieval in a steamer at 95 °C for 20 mins in Sigma Citrate Buffer, pH6.0, 10× (Sigma Aldrich, St. Louis, MO). To block endogenous peroxidase activity, slides were treated with a 3% hydrogen peroxide and rinsed in distilled water. The tissue sections were processed for IHC using the Thermo Autostainer 360 (ThermoFisher, Kalamazoo, MI). Sequential 15 min incubations with avidin D and biotin solutions (Vector, Burlingame, CA) were performed to block endogenous biotin reactivity. Specific anti-CCHFV immunoreactivity was detected using a primary polyclonal rabbit-α-CCHFV-NP antibody (IBT BioServices, Rockville, MD) at a 1:3200 dilution for 60 mins. A secondary biotinylated goat-α-rabbit-IgG (Vector Laboratories, Burlingame, CA) at 1:200 dilution for 30 mins followed by Vector Horseradish Peroxidase Streptavidin, R.T.U (Vector) for 30 mins. Slides were developed with Dako DAB chromagen (Dako, Carpenteria, CA) for 5 mins and counterstained with Harris hematoxylin for 30 seconds. Tissue sections from uninfected mice were used as negative controls.
Sigma citrate buffer
Manufactured by Merck Group
Sigma Citrate Buffer is a lab equipment product that provides a buffering solution. It maintains a specific pH range to support various laboratory applications and experiments.
Automatically generated - may contain errors
Lab products found in correlation
Thermo autostainer 360, by Thermo Fisher Scientific (3 mentions)
Vector horseradish peroxidase streptavidin, by Vector Laboratories (2 mentions)
Avidin d and biotin solutions, by Vector Laboratories (2 mentions)
Biotinylated goat αrabbit igg, by Vector Laboratories (1 mentions)
Dako dab chromagen, by Agilent Technologies (1 mentions)
Dako dab chromogen, by Agilent Technologies (1 mentions)
Goat anti rabbit igg, by Vector Laboratories (1 mentions)
3 protocols using sigma citrate buffer
1
Immunohistochemical Detection of CCHFV
2
Immunohistochemical Analysis of BDBV
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Necropsy was performed on all subjects in the BSL-4 facility. Tissue samples for histopathologic and immunohistochemical (IHC) examination were immersed in 10% neutral buffered formalin for at least 21 days, followed by a change of formalin, before removal from the BSL-4 laboratory. Inactivated tissue samples were processed in a BSL-1 laboratory. Tissue sections were deparaffinized and rehydrated through xylene and graded ethanols. Slides went through heat antigen retrieval in a steamer at 95°C for 20 minutes in Sigma citrate buffer, pH 6.0, 10 × (Sigma Aldrich). The tissue sections were processed for IHC using the Thermo Autostainer 360 (ThermoFisher). Specific anti-BDBV immunoreactivity was detected using an anti-BDBV GP primary antibody at a 1:2000 dilution for 60 minutes (IBT BioServices). Secondary antibody used was biotinylated goat anti-rabbit IgG (No. BA-1000; Vector Laboratories) at 1:200 for 30 minutes followed by Vector Streptavidin Alkaline Phosphatase at a dilution of 1:200 for 20 minutes (No. SA-5100; Vector Laboratories). Slides were developed with Bio-Red (No. BP-100-FR; Biopath Laboratories) for 7 minutes and counterstained with hematoxylin for 45 seconds.
3
Immunohistochemical Staining for Nipah Virus
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Tissue sections were deparaffinized and rehydrated through xylene and graded ethanol washes. Slides went through heat antigen retrieval in a steamer at 95°C for 20 minutes in Sigma Citrate Buffer, pH 6.0, 10× (Sigma-Aldrich). To block endogenous peroxidase activity, slides were treated with 3% hydrogen peroxide and rinsed in distilled water. The tissue sections were processed for IHC using the Thermo Autostainer 360 (Thermo Fisher Scientific). Sequential 15-minute incubations with avidin D and biotin solutions (Vector Laboratories, SP-2001) were performed to block endogenous biotin reactivity. Specific anti-NiV immunoreactivity was detected using an anti-NiV m102.4 human monoclonal antibody (53 (link)) at a 1:4,000 dilution for 60 minutes. Secondary antibody was biotinylated goat anti-rabbit IgG (Vector Laboratories, BA-1000) at 1:200 for 30 minutes followed by Vector Horseradish Peroxidase Streptavidin (ready-to-use; Vector Laboratories, SA-5704) for 30 minutes. Slides were developed with Dako DAB chromogen (Dako, K3468) for 5 minutes and counterstained with hematoxylin for 45 seconds.
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