Quantitative Immunofluorescence Analysis of α-SMA and MMP-9

For α-SMA and MMP-9 staining, FFPE blocks were sectioned, deparaffinised and rehydrated as described previously^{29 (link)}. Antigen retrieval was performed by incubating the sections in boiling citrate buffer (pH 6) for 45 minutes and cooled down at room temperature for 25 minutes. α-SMA antibody (Abcam, UK, ab7817, 1/50), MMP-9 (Santa Cruz Biotechnology, sc-10737, 1/50) and secondary goat anti-mouse AlexaFluor 488 (Life Technologies, Paisley, UK; A-11029, 1/200) and goat anti-rabbit AlexaFluor 546 (Life Technologies, Paisley, UK; A-11003, 1/200). Antibodies were used following the abcam TBS based staining protocol (https://www.abcam.com/protocols/immunostaining-paraffin-frozen-free-floating-protocol). Stained sections were mounted with ProLong® Gold Antifade with DAPI (Life Technologies, USA). Images from immunofluorescence staining were acquired using a Nikon Ti-e microscope. Mean fluorescence intensity was quantified using Fiji software by measuring the mean grey value in the MMP-9 channel for the randomly selected regions showing α-SMA positive staining. The values obtained from the different regions of the same tissue sections were averaged and treated as one experimental replicate. The final result was calculated for n = 20 HCC and 10 healthy patients per condition.