Vectra 3

Tumors fixed in 4% formaldehyde were transferred to 70% ethanol and embedded in paraffin at the Pathology Shared Resource–Research Histology, CU-AMC. Three tumors from each experimental group were sectioned, stained with DAPI and Ki67 primary antibody using Akoya Opal technology (Akoya Biosciences), and scanned at six representative regions with Vectra 3.0 (Akoya Biosciences) at the Human Immune Monitoring Shared Resource (CU-AMC). Next, InForm image analysis software (Akoya Biosciences) was used for automated identification of nuclei based on the DAPI signal. Ki67 nuclear fluorescence was outputted for all nuclei. Resulting libraries were downsampled based on power analysis calculation¹⁰⁹ using the formula n = (Zσ/E)² where n is the sample size required to ensure that the margin of error (E, 95%) does not exceed the value specified as 25% of the vehicle-treated nuclei signal, Z is the value from the table of probabilities of the standard normal distribution for the desired confidence level, and σ is the standard deviation of the outcome of interest. For antibody information see Supplementary Data 3.

Tissues with paraformaldehyde-fixed and paraffin-embedded were cut into 2 µm sections. After deparaffinized and rehydrated, the slices were fixed by 4% paraformaldehyde for 20 min and then antigen retrieval as normal IHC. mIHC staining was performed by using the Opal 7-Color Manual IHC Kit (NEL811001KT, Akoya Biosciences). The tumor sections were blocked in Opal Antibody Diluent/Block for 12 min at room temperature. Primary antibodies were incubated for 1 h at 37 °C or overnight at 4 °C. Following washing in TBST, the sections were incubated with secondary Abs Opal Polymer HRP Ms+Rb for 10 min at 37 °C. Sections were then washed in TBST and stained for 10 min with fluorescence staining diluted 1:100 in 1× Plus Amplification Diluent. All the slides were stained with DAPI for 5 min at room temperature and imaged by Akoya Vectra3. The images were sequentially spectrally unmixed by Akoya phenoptics inForm software.

Immunohistochemical staining was performed as described earlier on the Ventana Ultra Instrument (Ventana Medical Systems, Arizona, United States of America) [74 (link)]. Acquisition of the multispectral images was performed with the Vectra 3 automated imaging system (Akoya Biosciences). Data analysis was done in Imaris 9 software.

Immunohistochemical staining was conducted as in prior reports (20 (link)). Briefly, 4% paraformaldehyde (PFA) was used to fix brain tissues overnight before paraffin embedding. Next, serial 4-μm thick cerebral cortex sections were prepared, deparaffinized, rehydrated, and subjected to 0.01 M citric acid treatment for 10 min in a microwave at 400 W. Sections were treated for 12 min to block endogenous peroxidase activity, blocked for 30 min with bovine serum albumin (BSA), and treated overnight with polyclonal antibodies specific for VEGF (Beyotime Biotechnology, 1:200), CD31 (Abcam, 1:50), and NeuN (Sigma, 1:100) at 4°C. Sections were then washed and probed using an HRP-conjugated secondary antibody (Beyotime Biotechnology) for 1 h at 37°C, after which samples were evaluated via light microscopy (Perkin Elmer, USA). Integrated absorbance values were calculated by Vectra 3 (Akoya Biosciences, USA) to determine the positive protein expression area as follows: integrated absorbance = positive area × average absorbance.

Immunofluorescence staining was performed as previously described [27 (link),29 (link),30 (link)]. The sectioned tissues were deparaffinized in a dry oven at 60 °C and then rehydrated. The tissues were immersed to prevent nonspecific binding with normal goat serum (Jackson ImmunoResearch, West Grove, PA, USA) for 1 h at room temperature and incubated with primary antibodies using anti-Iba1 (1:100; Abcam, Cambridge, UK) and antineuronal nuclear (1:100; Millipore, Burlington, MA, USA) overnight at 4 °C. The following day, the incubated tissues were rinsed thrice using 1× phosphate-buffered saline with 0.01% Tween 20 (PBST) (Sigma-Aldrich, St. Louis, MO, USA) for 5 min, and then fluorescent secondary antibodies were treated with AlexaFluor-488 goat antirabbit (1:500; Invitrogen, Waltham, MA, USA) and AlexaFluor-594 goat antimouse (1:500; Invitrogen, Waltham, MA, USA) for 1 h at room temperature. After incubation, the tissues were rinsed thrice with 1× PBST for 5 min and dehydrated. Finally, the tissues were cover-slipped using a mounting medium containing 4′,6-diamidino-2-phenylindole (DAPI; Abcam, Cambridge, UK). Images were acquired using Vectra 3 (Akoya Bioscience, Marlborough, MA, USA) under a microscope at a magnification of 200×.