title: Development of positive control tissue for in situ hybridisation using Alvetex scaffolds.
abstract: BACKGROUND
  In situ hybridisation (ISH) is a robust method to determine the presence of mRNA for specific genes within a tissue. Ideally, positive and negative control tissues are used to determine probe specificity. However, this is not always possible, particularly for human genes where no knock-out controls exist.
  
  
  METHODS
  Here we report a novel method of growing positive control cells in a scaffold (Alvetex) to create 3D tissues suitable for sectioning with a cryostat. Sectioning slices through cells, similar to the effect on tissue and therefore provides improved penetration of the in situ riboprobes.
  
  
  METHODS
  ISH conducted on cells has been problematic due to the difficulty of efficient probe penetration, due to a semi-intact cell membrane, and cell preparations failing to withstand high stringency washes. Our new method circumvents this issue by enabling the positive control cells to be sectioned like a tissue.
  
  
  RESULTS
  HEK cells transfected with the genes of interest (in this case CB1 and NeuN) grown in Alvetex and cryosectioned were utilised to validate riboprobes and establish stringency conditions. These conditions were then transferred directly to human brain sections.
  
  
  CONCLUSIONS
  This method can be adapted to generate positive controls for ISH for any gene of interest. It provides a valuable option in human neuroscience where access to precious brain tissue is limited or where expression of a target gene is unknown.
authors.affiliation: Department of Pharmacology & Centre for Brain Research, School of Medical Sciences, University of Auckland, Faculty of Medical and Health Sciences, Private Bag 92019, Auckland 1142, New Zealand.
authors.forename: Christa
authors.initials: C
authors.lastname: MacDonald
authors.affiliation: Department of Pharmacology & Centre for Brain Research, School of Medical Sciences, University of Auckland, Faculty of Medical and Health Sciences, Private Bag 92019, Auckland 1142, New Zealand.
authors.forename: David Benjamin
authors.initials: DB
authors.lastname: Finlay
authors.affiliation: Department of Molecular Medicine, University of Auckland, Faculty of Medical and Health Sciences, Private Bag 92019, Auckland 1142, New Zealand.
authors.forename: Anower
authors.initials: A
authors.lastname: Jabed
authors.affiliation: Department of Pharmacology & Centre for Brain Research, School of Medical Sciences, University of Auckland, Faculty of Medical and Health Sciences, Private Bag 92019, Auckland 1142, New Zealand.
authors.forename: Michelle
authors.initials: M
authors.lastname: Glass
authors.affiliation: Department of Pharmacology & Centre for Brain Research, School of Medical Sciences, University of Auckland, Faculty of Medical and Health Sciences, Private Bag 92019, Auckland 1142, New Zealand. Electronic address: s.graham@auckland.ac.nz.
authors.forename: E Scott
authors.initials: ES
authors.lastname: Graham
mesh_terms: D000199:Actins / Q000378:metabolism; D000368:Aged; D034961:Antigens, Nuclear / Q000235:genetics / Q000378:metabolism; D002531:Cerebellum / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D007150:Immunohistochemistry; D017403:In Situ Hybridization / Q000295:instrumentation / Q000379:methods; D008297:Male; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism; D043884:Receptor, Cannabinoid, CB1 / Q000235:genetics / Q000378:metabolism; D046509:Tissue Culture Techniques / Q000295:instrumentation / Q000379:methods; D054457:Tissue Scaffolds; D014162:Transfection
chemical_list: C541116:ACTA2 protein, human; D000199:Actins; D034961:Antigens, Nuclear; D009419:Nerve Tissue Proteins; D043884:Receptor, Cannabinoid, CB1; C506808:neuronal nuclear antigen NeuN, human
keywords: Alvetex; Human; In situ hybridisation; Positive control; Tissue