Immunohistochemical analysis of paraffin-embedded SARS-CoV-2 positive human placenta using SARS-CoV-1/2 Spike Protein (2B3E5) Mouse mAb.
Immunohistochemical analysis of paraffin-embedded SARS-CoV-2 positive human placenta (top) or SARS-CoV-2 positive human lung (bottom) using SARS-CoV-1/2 Spike Protein (2B3E5) Mouse mAb (left) compared to SARS-CoV-2 Nucleocapsid Protein (E8R1L) Mouse mAb #33717 (right). The differential signal observed between SARS-CoV-1/2 Spike Protein (2B3E5) Mouse mAb and SARS-CoV-2 Nucleocapsid Protein (E8R1L) Mouse mAb #33717 antibodies is likely related to differences in antigen abundance of spike and nucleocapsid proteins.
Immunohistochemical analysis of paraffin-embedded SARS-CoV-2-infected hACE2 K18 mouse lung using SARS-CoV-1/2 Spike Protein (2B3E5) Mouse mAb (left) compared to secondary-only negative control (right). Note the mouse on mouse background staining in the absence of primary antibody. Human ACE2 transgenic mouse lung tissue was generously provided by Dr. Nicholas Crossland and Dr. Florian Douam, National Emerging Infectious Diseases Laboratories (NEIDL), Boston University.
Immunohistochemical analysis of paraffin-embedded 293T cell pellet transfected with SARS-CoV-2 spike protein (left-top) and various paraffin-embedded human tissues: colon adenocarcinoma (right-top), squamous cell lung carcinoma (right-bottom), and ductal breast carcinoma (left-bottom) using SARS-CoV-1/2 Spike Protein (2B3E5) Mouse mAb. Note the lack of staining in the tissues, all of which were procured prior to 2019, and therefore serve as reliable negative controls.
Immunohistochemical analysis of paraffin-embedded 293T cell pellet, untransfected (left) or SARS-CoV-2 spike protein transfected (right), using SARS-CoV-1/2 Spike Protein (2B3E5) Mouse mAb.
Confocal immunofluorescent analysis of HCT 116 cells transiently transfected with SARS-CoV-2 spike protein (left, positive) or mock transfected (right, negative), using SARS-CoV-1/2 Spike Protein (2B3E5) Mouse mAb (green), β-Actin (13E5) Rabbit mAb #4970 (red), and DAPI #4083 (blue).
|Immunohistochemistry (Paraffin)||1:200 - 1:800|
|Immunofluorescence (Immunocytochemistry)||1:200 - 1:800|
Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
NOTE: Do not allow slides to dry at any time during this procedure.
For Citrate: Heat slides in a microwave submersed in 1X citrate unmasking solution until boiling is initiated; follow with 10 min at a sub-boiling temperature (95°-98°C). Cool slides on bench top for 30 min.
|SignalStain® Boost IHC Detection Reagent (HRP, Mouse) #8125||SignalStain® Boost IHC Detection Reagent (AP, Mouse) #31926|
|SignalStain® DAB Substrate Kit #8059||SignalStain® Vibrant Red Alkaline Phosphatase Substrate Kit #76713|
|SignalStain® Vivid Purple Peroxidase Substrate Kit #96632|
NOTE: Use of detection reagents other than those specified in this protocol may require further optimization of the primary antibody to account for the different sensitivities of the detection reagents.
posted February 2010
revised June 2020
Protocol Id: 280
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalently purified water.
Recommended Fluorochrome-conjugated Anti-Mouse secondary antibodies:
NOTE: Cells should be grown, treated, fixed and stained directly in multi-well plates, chamber slides or on coverslips.
NOTE: All subsequent incubations should be carried out at room temperature unless otherwise noted in a humid light-tight box or covered dish/plate to prevent drying and fluorochrome fading.
posted December 2010
Protocol Id: 145
SARS-CoV-1/2 Spike Protein (2B3E5) Mouse mAb recognizes endogenous levels of total SARS-CoV-1 and SARS-CoV-2 spike protein.
Monoclonal antibody is produced by immunizing animals with SARS spike recombinant protein.
The cause of the COVID-19 pandemic is a novel and highly pathogenic coronavirus, termed SARS-CoV-2 (severe acute respiratory syndrome coronavirus-2). SARS-CoV-2 is a member of the Coronaviridae family of viruses (1). The genome of SARS-CoV-2 is similar to other coronaviruses, and is comprised of four key structural proteins: S, the spike protein, E, the envelope protein, M, the membrane protein, and N, the nucleocapsid protein (2). Coronavirus spike proteins are class I fusion proteins and harbor an ectodomain, a transmembrane domain, and an intracellular tail (3,4). The highly glycosylated ectodomain projects from the viral envelope surface and facilitates attachment and fusion with the host cell plasma membrane. The ectodomain can be further subdivided into host receptor-binding domain (RBD) (S1) and membrane-fusion (S2) subunits, which are produced upon proteolysis by host proteases at S1/S2 and S2’ sites. S1 and S2 subunits remain associated after cleavage and assemble into crown-like homotrimers (2,4). In humans, both SARS-CoV and SARS-CoV-2 spike proteins utilize the angiotensin-converting enzyme 2 (ACE2) protein as a receptor for cellular entry (5-7). Spike protein subunits represent a key antigenic feature of coronavirus virions, and therefore represent an important target of vaccines, novel therapeutic antibodies, and small-molecule inhibitors (8,9).
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