Protocol detail

Characterization of Immune Infiltration in OPSCC

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Characterization of lymphocytic infiltration was carried out with triple immunofluorescent staining in 41 OPSCC as described previously [10 (link)] using anti-CD8 (mouse IgG2b, clone 4B11; Novocastra, 1:400), anti-Tbet (rabbit polyclonal, clone H210; Santa Cruz 1:400) and anti-Foxp3 (mouse IgG1, clone 236A/E7; Abcam, 1:200), goat-anti-mouse IgG2b Alexa 647, goat-anti-rabbit Alexa 546 and goat-anti-mouse IgG1 Alexa 488 (all from Molecular probes; 1:200). Based on the morphology of cancer cell nests and autofluorescence of keratinocytes the immune cells per mm² were manually counted as intraepithelial or stromal using the LSM 5 Image Examiner software (average of five images at a 250× magnification).

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Santegoets S.J., Duurland C.L., Jordanova E.S., van Ham J.J., Ehsan I., van Egmond S.L., Welters M.J, & van der Burg S.H. (2019). Tbet-positive regulatory T cells accumulate in oropharyngeal cancers with ongoing tumor-specific type 1 T cell responses. Journal for Immunotherapy of Cancer, 7, 14.

Publication 2019

anti-Tbet anti-Foxp3 goat-anti-mouse IgG2b Alexa 647 goat-anti-rabbit Alexa 546 goat-anti-mouse IgG1 Alexa 488

Based cell cancer Clone Goat Igg1 Igg2b Immunofluorescent Keratinocytes Lymphocytic Molecular probes Mouse Rabbit

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Variable analysis

independent variables

Immunofluorescent staining methods: anti-CD8, anti-Tbet, anti-Foxp3 antibodies

dependent variables

Density of intraepithelial and stromal immune cells per mm^2 in OPSCC samples

control variables

OPSCC samples (n=41)
Imaging using LSM 5 Image Examiner software at 250x magnification
Manual counting of immune cells based on morphology and autofluorescence

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

Annotations

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