Microbeam 4

Serial 8μm cryosections (Leica CM3050S, UK) of OCT-embedded keloid and NS samples were cut onto specialised polyethylene naphthalate (PEN) membrane slides (Carl Zeiss, UK). To differentiate epidermis from dermis, whilst preserving tissue RNA integrity, a rapid staining protocol was performed (LCM Staining Kit, Ambion, Austin TX, USA) according to the manufacturer’s instructions [16 (link), 17 (link)]. Using a P.A.L.M. LCM microscope (Carl Zeiss MicroBeam 4.2, Germany) epidermis and dermis of each sample was laser cut and catapulted away from the slide into separate overhanging microtube caps (AdhesiveCap 200 Opaque, Carl Zeiss Microscopy Ltd, Cambridge, UK). Multiple ‘elements’ were captured from each tissue section of least three sequential sections from each patient, ensuring adequate biological representation. The captured tissue was mixed with lysis buffer (Buffer RLT with 1% 2-mercaptoethanol, RNeasy Micro Kit, Qiagen, UK) and stored at -80°C until extraction according to manufacturer’s instructions (RNeasy Micro Kit, Qiagen, UK). Following extraction, the samples were again stored at -80°C [18 (link)].