Unstained formalin-fixed, paraffin-embedded (FFPE) human cerebral meningeal 5 μm sections were obtained from Amsbio. For antigen retrieval, slides were incubated in pH 6.0 buffer (Reveal Decloaking reagent, Biocare Medical) at 95–98°C for 30 minutes followed by a cool down period. Subsequent steps were then performed using an immunohistochemical staining platform (Intellipath, Biocare). Slides were immersed in a 3% hydrogen peroxide solution (Peroxidazed, Biocare) for 10 minutes, rinsed with TBST and blocked using a serum-free solution (Background Sniper, Biocare Medical, Concord, CA) for 10 minutes. The blocking solution was removed and the slides were incubated with human CXCL12/SDF-1 antibody (Clone D8G6H; Cell Signaling Technology) or IgG isotype control antibody (Cell Signaling Technology, #3900) 2.7 μg/mL in 10% blocking solution/90% TBST for 60 minutes at room temperature. Slides were then washed, and detection was performed using the Novocastra Novolink Polymer Kit (Leica Microsystems Inc.) according to the manufacturer’s specifications. Detection was performed with diaminobenzidine (DAB; Biolegend). Finally, the slides were counterstained with CAT hematoxylin (Biocare), dehydrated, and coverslipped.
Igg isotype control antibody
Manufactured by Cell Signaling Technology
The IgG isotype control antibody is a laboratory tool used to establish background staining levels in immunoassays. It serves as a reference point to distinguish specific antigen-antibody interactions from non-specific binding.
Automatically generated - may contain errors
Lab products found in correlation
Background sniper, by Biocare Medical (1 mentions)
Intellipath, by Biocare Medical (1 mentions)
Peroxidazed, by Biocare Medical (1 mentions)
Reveal decloaking reagent, by Biocare Medical (1 mentions)
Novocastra novolink polymer kit, by Leica (1 mentions)
Diaminobenzidine dab, by BioLegend (1 mentions)
Cat hematoxylin, by Biocare Medical (1 mentions)
Avidin biotin blocking kit, by Vector Laboratories (1 mentions)
Dapi 4 6 diamidino 2 phenylindole, by Vector Laboratories (1 mentions)
Fluorescence microscope, by Zeiss (1 mentions)
Vectashield mounting medium, by Vector Laboratories (1 mentions)
2 protocols using igg isotype control antibody
1
Immunohistochemical Analysis of CXCL12/SDF-1 in FFPE Meninges
2
Visualization of Ganglioside GM1 and CMIP in T Cells
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T cells were plated at 104 cells/slide using a cytospin centrifuge, fixed in 4% PFA for 10 min at RT, and then permeabilized by incubation with 0.3% Triton X-100 for 10 min. Endogenous biotin and avidin were blocked using an Avidin-Biotin Blocking kit (Vector Laboratories). Membranous ganglioside GM1 was visualized by direct incubation with Alexa Fluor555-conjugated cholera toxin subunit B (Molecular Probes, 8 µg/ml in PBS, 50 µl/slide) for 25 min at RT and washed (3 × 5 min) before being covered with Vectashield mounting medium containing DAPI (4′,6-diamidino-2-phenylindole) (Vector Laboratories), following which the slides were viewed under a fluorescence microscope (Zeiss, Germany) with the appropriate filters. A rabbit polyclonal antibody developed in our laboratory was used (1:1000 dilution) to visualize CMIP. An anti-CMIP antibody recognizing both human and mouse CMIP has been previously described.17 (link) To ensure the specificity of each signal, IgG isotype control antibody (Cell Signaling) was used instead of primary antibody in control samples.
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