The abundance of TITreg cells was analyzed utilizing Opal 7-Colour Manual IHC Kit (PerkinElmer NEL811001KT) according to the manufacture’s protocol (14 (link)). In brief, the slides were incubated with Antibody Diluent blocking buffer (PerkinElmer) at room temperature (RT) for 10 min. The primary antibody for CD4 (Abcam, ab133616, 1:500) and FoxP3 (R&D, MAB8214, 1:400) were incubated at RT for 1 h. Then, a secondary HRP antibody were incubated at RT for 10 min. Signal amplification was performed using Opal 520 TSA (PerkinElmer) and incubated at RT for 10 min. Visualization of the slides was done using the Mantra Quantitative Pathology Imaging System (PerkinElmer) and analyzed using InForm Image Analysis software (PerkinElmer, version 2.1). The TITreg cell fraction in CD4+ cells were calculated and data were presented as mean ± SD.
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Quantitative Analysis of Tumor-Infiltrating Treg Cells
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Opal 7-Colour Manual IHC Kit
Antibody Diluent blocking buffer
MAB8214
Mantra Quantitative Pathology Imaging System
InForm Image Analysis software
Antibody
Blocking antibody
Buffer
Cd4 cells
Opal
Corresponding Organization : Tianjin Medical University Cancer Institute and Hospital
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Variable analysis
independent variables
- Antibody Diluent blocking buffer (PerkinElmer)
- Primary antibody for CD4 (Abcam, ab133616, 1:500)
- Primary antibody for FoxP3 (R&D, MAB8214, 1:400)
- Secondary HRP antibody
- Opal 520 TSA (PerkinElmer)
- The TITreg cell fraction in CD4+ cells
- Incubation time and temperature for each step
- No positive or negative controls were explicitly mentioned.
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