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. 2023 Jan 29;15(3):827. doi: 10.3390/cancers15030827

Figure 4.

Figure 4

Amplification strand (AmpS) heating. (A) MCF7-HER2 xenograft FFPE tissue sections were stained with either a HER2 primary antibody for indirect IF or a HER2 Ab-oligo conjugate (HER2 = yellow and DAPI = white), where AmpS was either kept at room temperature prior to application or heated to 85 °C prior to staining. (B) Negative controls for each sample were stained with only a fluorophore-labeled secondary antibody or the respective AmpS staining conditions and equivalent Amp IS concentrations. (C) Image quantification was performed to calculate the HER2 signal-to-background ratio (SBR) for indirect IF and the Ab-oligo amplification strategy with and without heating of the AmpS prior to staining. (D) Indirect IF was compared to the Ab-oligo amplification strategy (20× Amp S to AmpS) using 85 °C heating across antigens including HER2, alpha-smooth muscle actin (α-SMA), CD4, cytokeratin 8 (CK8), CoxIV, CD68, PD1, PCNA, CK7, CK14, androgen receptor (AR), Vimentin (VIM), estrogen receptor (ER), progesterone receptor (PR), and E-Cadherin (E-Cad). Images shown are representative of multiple fields of view and quantification results are reported as a representative average of all images. Scale bar = 50 μm.