Opal 4 color kit

Tumors were fixed overnight in 4% paraformaldehyde, transferred to 70% EtOH and paraffin-embedded. Sections 4µm-thick were deparaffinized, rehydrated and incubated with anti-CD31 (1:100, DIA 310, Clinisciences) followed by goat-anti rat 594 (10348312, Life Technology) and/or with anti- alpha-smooth muscle actin (α-SMA)-Cy3 (1: 250, C6198, Sigma) antibody at room temperature during 1 hour.
Alternatively, sections were incubated successively with GLUT1 (1:1000, ab115730, Abcam) and anti-CD31 (1:600 DIA 310, Clinisciences) using the OPAL 4 color kit (NEL820001KT, PerkinElmer) at room temperature during 1 hour. The secondary antibody was Opal Polymer anti-rabbit HRP (ARR1001KT, PerkinElmer) for GLUT1 and anti-rat HRP (MP-7444-15, Eurobio) for CD31.
Nuclei were stained with DAPI and slides were coverslipped and scanned with Vectra^® Polaris^™ (PerkinElmer). Analysis of immunofluorescence was performed with homemade software developed in Matlab® (The MathWorks, Natick, MA, USA). CD31, GLUT1 and α-SMA signals were counted in 20 fields of 3 independent sections at x20 magnification for each sample. Pericyte coverage was calculated as the number of vessel covered by α-SMA (both α-SMA and CD31 positives vessels) over the total number of CD31 positive vessels.

Serial paraffin sections (4 µm) from human CSCC tissues were analyzed by immunofluorescence with the Opal 4-Color Kit (PerkinElmer) according to the manufacturer’s protocol. After deparaffinization, sections were microwaved in antigen retrieval buffer for 45 s at 100 °C, washed and blocked for 10 min at room temperature, followed by incubation with anti-VASH1 antibody (ab176114, Abcam). Horseradish peroxidase-conjugated secondary antibody was dropped onto slides for incubation for 10 min at room temperature. Subsequently, tyramide signal amplification (TSA) working buffer (Opal 570) was used to amplify the signal on slides. After eliminating anti-VASH1 and secondary antibodies by microwaving, the above procedures were repeated with anti-LYVE1 antibody (ab33682, Abcam) and TSA working buffer (Opal 520). Sections were mounted in neutral gum and visualized by a fluorescence microscope (Olympus). The fluorescence intensity of VASH1 expression was analyzed by ImageJ software.

Serial paraffin sections (4 μm) from human cervical cancer tissues were analysed by immunofluorescence with an Opal 4-Color Kit (PerkinElmer) according to the manufacturer’s protocol^{15 (link)}. Briefly, sections were microwaved in antigen retrieval buffer for 15 min at 90 °C after deparaffinization; then, they were washed and blocked for 10 min at room temperature and incubated with an anti-ZEB1 antibody. Horseradish peroxidase-conjugated secondary antibody was dropped onto slides for incubation for 10 min at room temperature. Subsequently, tyramide signal amplification (TSA) buffer (Opal 650) was used to amplify the signal on the slides. After eliminating the anti-ZEB1 and secondary antibodies by microwaving, the above procedures were repeated with an anti-CAIX antibody and TSA buffer (Opal 570). The above procedures were repeated again with an anti-CD163 antibody (ab156769; Abcam) and TSA buffer (Opal 520). The sections were mounted in neutral gum and visualized by a fluorescence microscope (Olympus).