Lmd7000 system

Eight formalin-fixed paraffine-embedded samples were used for laser microdissection, 4 from non-metastatic cases and 4 from metastatic cases. Eight-μm sections from each sample were mounted onto plastic membrane slides (Leica Microsystems, Germany), stained with hematoxylin-eosin, air dried and stored at -80°C until use. Laser microdissection was performed with the Leica LMD7000 System (Leica). Approximately 3 mm² of either tumoral epithelium or stroma were collected separately for each sample. RNA isolation was performed with the RNeasy FFPE Kit (Qiagen) and reverse transcribed with the High-Capacity cDNA Reverse Transcription Kit (Life Technologies). cDNA was preamplified (14 cycles) with the TaqMan PreAmp Master Mix (Life Technologies). SPARC transcript levels were quantified using a TaqMan assay (Hs00234160_m1) and normalized to β-2 microglobulin (B2M) transcript levels (assay Hs00984230_m1). Real-time PCR assays were performed and analyzed as described above.

A total of 44 frozen and 127 paraffin-embedded cervical/vulvar specimens were retrieved from the Tissue Biobank of the University Hospital Center of Liege (Belgium). All patients underwent a surgical procedure between 2011 and 2015. An informed consent was obtained from all participants and the protocol was approved by the local ethics committee. These tissue samples included 31 normal squamous tissues (ectocervix/TZ), 65 dysplastic lesions (30 LSIL and 35 HSIL) and 75 primary invasive squamous cell carcinomas (SCC) (33 cervical and 42 vulvar cancers). The original diagnoses were re-examined by senior histopathologists and, to avoid misclassification, both the proliferative index (nuclear Ki67 staining) and p16^INK4a expression of each specimen were determined by immunohistochemistry. In order to perform our transcriptional analyses on “pure” populations of epithelial cells (and to avoid any contamination with stromal structures), several serial sections (6 μm thick) of each frozen cervical specimen were microdissected using a Leica LMD7000 system (Leica, Wetzlar, Germany) (GIGA in vitro Imaging platform, University of Liege). To obtain sufficient RNA concentrations, microdissected squamous cell populations from different slides but from the same tissue sample were pooled.

Mice were euthanized 24 hours post RD, eyes were enucleated, embedded in O.C.T. compound (Tissue Tek; Sakura Finetek, Torrance, CA, USA) and fresh-frozen at −80 °C. Eyes were then cut in the sagittal plane at 20 µm-thickness on a cryostat (Leica CM1850; Leica Biosystems, Buffalo Grove, IL, USA) and serial sections were collected on polyethylene terephthalate-membrane (PET) frame slides (PET FrameSlide #0010; steel frames, RNase-free, material number #11505190, Leica Microsystems, Wetzlar, Germany). Sections were fixed in 75% ethanol (30 seconds), washed with nuclease-free water (30 seconds), stained with 0.02% toluidine blue solution for 20 seconds and washed again as described above. Finally, sections were dehydrated with 75%, 95% and 100% ethanol (30, 30 and 2 × 30 seconds respectively). LCM was performed with the Leica LMD7000 system and LMD application version 7.5 (Leica Microsystems, Wetzlar, Germany). Photoreceptors’ layer was cut by laser and collected into 0.5 ml tubes containing RNAlater stabilization solution (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA).

Following histological examination of H & E staining of oral mucosal tissues (i.e., NHOE (n = 4), precancerous oral lesions (n = 4) and OSCC tissues (n = 4)), epithelial layers from the paraffin-embedded tissue samples were excised by laser-captured microdissection (LCM) using Leica (LMD) 7000 system (Leica Microsystems Inc., Richmond, IL, USA) at the California NanoSystems Institute at UCLA (Los Angeles, CA, USA). LCM-derived tissue RNAs were extracted using a high pure RNA paraffin kit (Roche, Basel, Switzerland). RT was performed with RNA isolated from the tissue sections using a Superscript II RT kit (Invitrogen) with random hexamer primers (Promega, Madison, WI, USA) according to the manufacturer’s instructions. Then, qPCR was performed using PowerUp SYBR Green Master Mix (Thermo Fisher Scientific, Waltham, MA, USA) and QuantStudio 3 qPCR System (Thermo Fisher Scientific) as described in our prior work [32 (link)]. Thermocycling conditions for all PCR reactions included an initial denaturation stage at 95 °C for 10 min, followed by 50 cycles.