Ecomount

The experimental sequence is summarized in the following steps. Mice were humanely sacrificed after treatment. The tumor tissues were sectioned into 4 μm-thick sections for immunohistochemical analyses (IHC). The deparaffinization consisted in exposing the tissues to a temperature of 60 °C for 15–30 min, followed by rehydration. The samples were put in contact for 5 min with the following solutions: xilol (x2), ethanol/xylol 50:50, ethanol 100%, ethanol 96%, ethanol 80%, ethanol 70%, ethanol 50%, distilled water, and PBS. A solution of Triton x-100 0.5% was used to permeabilize the cell membrane (30 min), followed by 2 washes with PBS of 5 min each. Hematoxylin 100–200 μL was added to cover the sample and distilled water was used to wash the samples. Samples then were covered with eosin and finally assembled with EcoMount from Biocare Medical. Photomicrographs were taken from xenotransplant tissues of HCT116 cells stained with HE. We acquired 30 photomicrographs per experimental group. The acquisition of images was done through an Olympus IX71 inverted microscope using the Qcapturepro 5 software of the company QImaging at 20× of the microscopic scale.

Paraffin-embedded tissue samples were sectioned at
a thickness of
3–4 μm on charged microscope slides. Samples were deparaffinized
using CitriSolv (Decon Laboratories, Inc., King of Prussia, PA) and
rehydrated sequentially in graded alcohols. Antigen retrieval was
performed by incubating slides in 10 μg/mL proteinase K (MilliporeSigma)
in PK buffer (0.6 M Tris (pH 7.5)/0.1% CaCl₂) for 10 min
at RT. Blocking of endogenous peroxidase and alkaline phosphatase
was performed by incubating slides in Rodent Block M (BioCare Medical,
Pacheco, CA) followed by incubation with BLOXALL Endogenous Blocking
Solution (Vector Laboratories, Burlingame, CA). Slides were washed
in TBS buffer and incubated with primary rabbit Salmonella O Antiserum
(Group B Factors 1, 4, 5, 12, #BD 229481, Becton, Dickinson and Company)
for 1 h at 1:5000 dilution. Slides were then incubated in AP-polymer
(Rabbit on Rodent AP-Polymer, BioCare Medical) followed by Vina Green
Chromogen (BioCare Medical, Pacheco, CA) treatment. Tissues were finally
counterstained with hematoxylin (BioCare Medical) before mounting
with EcoMount (BioCare Medical). Slides were cured at 60–70
°C for 15 min.

We examined multiple ganglia (n = 32) along the entire neuraxis, from both SVV-wt- and SVV-EGFP-infected AGMs at 9 dpi [animals 269 (SVV-wt) and 294 (SVV-EGFP)], 13 dpi [animals 279 (SVV-wt) and 273 (SVV-EGFP)], and 20 dpi [animal 283 (SVV-EGFP)] (Table 1) by in situ hybridization (ISH), according to the manufacturer’s instructions (Advanced Cell Diagnostics). Briefly, deparaffinized 5-μm tissue sections were incubated with probes against SVV ORF63, CXCL10, ubiquitin C (positive control), and the bacterial DapB gene (negative control) using the RNAscope 2.0 Kit Red (Advanced Cell Diagnostics), as described (Ouwendijk et al. 2013a (link)). Signal was visualized using FastRed as a substrate, and slides were counterstained with hematoxylin and mounted in Ecomount (Biocare Medical). Each ISH staining was performed on one to two sections at different levels of the tissue.

Colorimetric in-situ hybridization (ISH) was performed manually on superfrost plus slides (Thomas Scientific, Swedesboro, NJ), according to the manufacturer’s instructions [72 (link)], using the RNAscope 2.5 HD Red Reagent Kit (Advanced Cell Diagnostics, Newark, CA) and RNAscope Probe V-ZIKVsph2015 (Advanced Cell Diagnostics). Briefly, each 5 μm section of formalin-fixed, paraffin embedded tissue was pretreated with heat and protease, followed by ZIKV probe (GenBank accession number KU321639.1 [complete genome]; 70 pairs; target region 130–4186) hybridization for 2 hours at 40°C, a cascade of signal amplification molecules, and signal detection. Slides were counterstained with hematoxylin and mounted with xylene based EcoMount (BioCare Medical, Pacheco, CA). A probe designed to detect bacterial dapB (Advanced Cell Diagnostics, Newark, CA) was used as a negative control. A section of spleen from a ZIKV-infected rhesus macaque euthanized 4 DPI was used as a positive control. ISH was only performed on tissues that tested positive via qRT-PCR and was not performed on samples that contained less than 5 log₁₀ genomes/mg tissue. Positive staining was identified as red cytoplasmic or perinuclear staining. When possible, ISH-positive cell types were identified by the pathologist based on tissue location and cell morphology.

C57BL/6J, 5-month-old mice were anesthetized with isoflurane and transcardially perfused first with PBS followed by 10% NBF. Brains were fixed for 24 hours at 4°C in 10% NBF and then switched to 30% sucrose for 24 hours. Brain slices (25 μm) were cut using a freezing microtome (Leica), collected in PBS and treated with hydrogen peroxide for 10 minutes. After a PBS rinse, slices were mounted and desiccated overnight at room temperature. In situ hybridization was performed using RNAScope multiplex fluorescence kits (cat# 323110) and Klb (cat# 415221 Mm-Klb) and Fgfr1c (cat# 454941-C2 Mm-Fgfr1-O1-C2) probes purchased from Advanced Cell Diagnostics. Hybridized slides were incubated with amplification reagents and Opal 570 and 690 dyes (Akoya Biosciences, 1:1500 dilution) followed by sequential incubation with tyrosine hydroxylase (Aves Labs, 1:1000 dilution), biotinylated anti-chicken secondary (Jackson ImmunoResearch, 1:1000 dilution) and streptavidin AlexaFluor488 (Invitrogen, 1:1000 dilution) antibodies. Slides were rinsed, dehydrated, cleared, and mounted with EcoMount (BioCare Medical). Images were taken using a Zeiss LSM880 confocal microscope.

For the in situ hybridization (ISH) assay, the RNAScope 2.5 HD Detection Reagent-Red kit (Advanced Cell Diagnostics, Inc., CA, United States) was used. The procedure was performed following the manufacturer’s instructions. Briefly, tissue sections were cleared and hydrated in xylene and 100% ethanol and then air-dried. The sections were quenched for 10 min in aqueous H₂O₂, boiled in target retrieval solution for 15 min, rinsed in 100% ethanol and air-dried again. Then a final pre-treatment of protease plus enzyme for 15 min at 40°C was applied. The V-AFSV-01 probe (Advanced Cell Diagnostics, Inc., CA, United States) was applied and incubated at 40°C for 2 h. Sections were then washed twice in 1× wash buffer. After each of the subsequent hybridization steps, the sections were washed twice again. The signal was visualized with the chromogen Fast Red. The sections were then counterstained with Gill’s 1 hematoxylin, dried, cover-slipped with EcoMount (BioCare Medical, CA, United States), and examined by the pathologist. A negative control lymph node from a non-infected animal was tested in an ISH assay, and no immunostaining was observed. In addition, a negative control probe on positive tissue (lymph node from an ASFV Georgia 2007/1 infected animal) was tested, and no staining was observed.