Palm microbeam instrument

Mice were culled and tissues were fixed in situ by perfusing the mice from the left ventricle with 0.5% paraformaldehyde and 20% sucrose made in nuclease free PBS (fixative). DRG (T8–T12) were harvested, immersed in the fixative, and allowed to equilibrate on ice. Fixed DRG from two mice were pooled and then embedded in OCT (TissueTekIA018; Sakura) and frozen in liquid nitrogen. Frozen DRG were sectioned into 8 µm slices on a cryostat (Leica CM3050S) and consecutive DRG sections were placed on each subsequent slide (LOMB) that has been pre-treated with RNaseAway (Life Technologies). Freshly prepared tissue sections were dehydrated with 50%, 75% and 95% ethanol sequentially and air-dried just before laser capture. YFP⁻ and YFP⁺ cells were identified from 8–24 non-consecutive tissue sections and laser microdissected using a PALM MicroBeam instrument (Carl Zeiss). Laser capture was performed at the Biological Optical Microscopy Platform, University of Melbourne, Australia. Cells were then captured into collection tubes containing lysis buffer. We avoided collecting cells from 2–9 consecutive sections to prevent repeated sampling from the same cell. In total, 205 YFP⁺ and 256 YFP⁻ cells from HSV infected DRG and 136 cells from uninfected DRG were collected from 3–4 independent experiments with two mice per OCT block.

Fresh frozen colon adenocarcinoma specimens were cut into 12 μm serial sections and mounted on PALM membrane slides (Zeiss, Oberkochen, Germany). The slides were immediately stained with cresyl violet from the LCM staining kit (Thermo Fischer Scientific) and laser capture microdissection (LCM) was performed immediately thereafter. Adenocarcinomatous and stromal areas were selected during the LCM procedure by a pathologist (CBR). Laser capture microdissection was performed with the PALM MicroBeam instrument (Zeiss). At least 5 mm² of tumor tissue or stromal tissue were collected from each sample. This required from nine to twelve 12 μm sections.
RNA from tumor and stromal microdissected tissues were isolated and purified with the RNeasy micro kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer’s instructions. RNA concentrations were measured using NanoDrop system (Thermo Fisher Scientific). RT-PCR analyses were performed as detailed above.