Immunohistochemistry (IHC) and immunofluorescence (IF) staining were performed at the MSK Molecular Cytology Core Facility using Discovery XT processor (Ventana Medical Systems) as previously described [22 (link)]. Tumor samples were fixed and embedded in paraffin. Anti-human CD45, anti-human CD3, anti-human CD4, anti-human CD8, anti-human FoxP3, anti-mouse CD45, anti-mouse CD11b, anti-mouse CD68, and anti-mouse IBA1 antibodies were used, which were followed by biotinylated secondary antibody. The detection was performed using a DAB detection kit (Ventana Medical Systems) or Alexa Fluor™ 488 or 568 Tyramide Reagent (Invitrogen). IHC images were captured from tumor sections using a Nikon ECLIPSE Ni-U microscope and NIS-Elements 4.0 imaging software. IF images were captured using Leica Inverted Confocal SP8 and processed with Imaris (Bitplane). Antigen positive cells were counted with Qupath 0.1.2.
Alexa fluor 488 or 568 tyramide reagent
Manufactured by Thermo Fisher Scientific
The Alexa Fluor™ 488 or 568 Tyramide Reagent is a fluorescent labeling reagent used in immunohistochemistry and in situ hybridization techniques. It is designed to provide sensitive and specific detection of target molecules in fixed and permeabilized samples.
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3 protocols using alexa fluor 488 or 568 tyramide reagent
1
Immunohistochemistry and Immunofluorescence of Tumor Samples
2
Immunohistochemistry and Immunofluorescence Imaging of Tumor Samples
3
Immunohistochemical Profiling of Tumor Immune Microenvironment
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Immunohistochemistry (IHC) staining was performed at the MSK Molecular Cytology Core Facility using Discovery XT processor (Ventana Medical Systems) as previously described.24 25 (link) Tumor samples were fixed and embedded in paraffin. Anti-human CD45 (Abcam Cat# ab10558, RRID:AB_442810), anti-human CD3 (Agilent, Cat# A0452, RRID:AB_2335677), anti-human CD4 (Ventana Medical Systems Cat# 790-4423, RRID:AB_2335982), anti-human CD8 (Ventana Medical Systems Cat# 790-4460, RRID:AB_2335985), and anti-mouse CD31(Abcam Cat# ab134168, RRID:AB_2890012) were used, followed by a biotinylated secondary antibody. The detection was performed using a DAB detection kit (Ventana Medical Systems) or Alexa Fluor 488 or 568 Tyramide Reagent (Invitrogen). IHC staining of peripheral node addressin (PNA) (MECA79) was done with a manual protocol at Laboratory of Comparative Pathology using anti-PNA antibody (MECA79R) (Novus Cat# NBP2-78792). Immunofluorescence (IF) double staining of PNA and human CD3 was performed with the identical anti-PNA antibody and monoclonal rabbit anti-human CD3 antibody (clone MRQ-39, LS-C202826). Microscopic images were captured from tumor sections using a Nikon ECLIPSE Ni-U microscope and NIS-Elements V.4.0 imaging software. Antigen positive cells were counted with Qupath V.0.1.2 or using positive pixel count analysis.
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