After chromogen-based IHC analysis was used for all of the targets, each target was assessed by a uniplex IF assay to optimize the antibodies and to generate spectral libraries required for multiplex IF image analysis. Uniplex IF staining was performed manually by using the Opal 7 kit (catalogue #NEL797001KT; PerkinElmer, Waltham, MA), which uses individual tyramide signal amplification (TSA)-conjugated fluorophores to detect various targets within an IF assay. After deparaffinization, slides were placed in a plastic container filled with antigen retrieval (AR) buffer in Tris-EDTA buffer (for CD4, CD3, granzyme B, and CD57 analysis) or citrate buffer (for analysis of the remaining markers); microwave technology (EZ-RETRIEVER® system microwave from BioGenex) was used to bring the liquid to the boiling point (1 min) at 100 °C, and the sections were then microwaved for an additional 15 min at 75 °C. Slides were allowed to cool in the AR buffer for 15 min at room temperature and were then rinsed with deionized water and 1 × Tris-buffered saline with Tween 20 (TBST; Santa Cruz Biotechnology, Dallas, TX). To initiate protein stabilization and background reduction, Tris-HCl buffer containing 0.1% Tween (Dako, catalogue #S3022) was used for 10 min at room temperature. Slides were then incubated between 30 min and 2 h (depending on which antibody was used at room temperature) with the same primary antibodies used for IHC analysis against the immune markers at specific dilutions: AE1/AE3 (dilution 1:300), PD-L1 (dilution 1:3000), CD4 (dilution 1:80), CD8 (dilution 1:120), CD3 (dilution 1:100), PD-1 (dilution 1:250), granzyme B (dilution 1:1), CD57 (dilution 1:10), CD45RO (dilution 1:1), FOXP3 (dilution 1:50), and CD68 (dilution 1:450). Next, the slides were washed and incubated for 10 min at room temperature with anti-mouse or anti-rabbit secondary antibodies (Novocastra, Leica Biosystems) after successive washes in TBST.
The slides were then incubated at room temperature for 10 min with one of the following Alexa Fluor tyramides (PerkinElmer) included in the Opal 7 kit to detect antibody staining, prepared according to the manufacturer’s instructions: Opal 520, Opal 540, Opal 570, Opal 620, Opal 650, and Opal 690 (dilution 1:50). After three additional washes in deionized water, the slides were counterstained with DAPI for 5 min and mounted with VECTASHIELD Hard Set (Vector Labs, Burlingame, CA). Autofluorescence (negative control) slides were also included, using primary and secondary antibodies and omitting the fluor tyramides. As performed with the IHC staining, the correct titration in the uniplex IF slides was chosen carefully to obtain a uniform, specific, and correct staining pattern. Similar to IHC validation, positive and negative controls were used during each run staining.
The slides were then incubated at room temperature for 10 min with one of the following Alexa Fluor tyramides (PerkinElmer) included in the Opal 7 kit to detect antibody staining, prepared according to the manufacturer’s instructions: Opal 520, Opal 540, Opal 570, Opal 620, Opal 650, and Opal 690 (dilution 1:50). After three additional washes in deionized water, the slides were counterstained with DAPI for 5 min and mounted with VECTASHIELD Hard Set (Vector Labs, Burlingame, CA). Autofluorescence (negative control) slides were also included, using primary and secondary antibodies and omitting the fluor tyramides. As performed with the IHC staining, the correct titration in the uniplex IF slides was chosen carefully to obtain a uniform, specific, and correct staining pattern. Similar to IHC validation, positive and negative controls were used during each run staining.
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