title: Automated Double In Situ Detection of Mouse Lgr5 mRNA and Lysozyme Protein in Examining the Neighboring Cell Types of the Mouse Intestinal Crypt.
abstract: Automated detection of mRNAs and proteins in the same tissue sections is not a routine procedure. Successful experiment depends on the preparation of the tissue, the detection procedure, as well as the quality of the probes and antibodies. The multiplexed detections require experimental conditions, preserving the state of the molecular targets of interest and providing expression pattern of each target the same as in a single detection. Here we describe in detail the automated protocols used to detect mouse Lgr5 mRNA by in situ hybridization and immunofluorescence detection of lysozyme in the same mouse intestinal sections. Both the in situ hybridization and the protein detection were performed with an automated staining processor and provided strong and reproducible results.
authors.affiliation: Molecular Cytology Core Facility, Sloan Kettering Institute for Cancer Research, Memorial Sloan Kettering Cancer Center, 1275 York Avenue, Box # 333, New York, NY, 10065, USA.
authors.forename: Mesruh
authors.initials: M
authors.lastname: Turkekul
authors.affiliation: Molecular Cytology Core Facility, Sloan Kettering Institute for Cancer Research, Memorial Sloan Kettering Cancer Center, 1275 York Avenue, Box # 333, New York, NY, 10065, USA.
authors.forename: Afsar
authors.initials: A
authors.lastname: Barlas
authors.affiliation: Molecular Cytology Core Facility, Sloan Kettering Institute for Cancer Research, Memorial Sloan Kettering Cancer Center, 1275 York Avenue, Box # 333, New York, NY, 10065, USA.
authors.forename: Dmitry
authors.initials: D
authors.lastname: Yarilin
authors.affiliation: Molecular Cytology Core Facility, Sloan Kettering Institute for Cancer Research, Memorial Sloan Kettering Cancer Center, 1275 York Avenue, Box # 333, New York, NY, 10065, USA.
authors.forename: Sho
authors.initials: S
authors.lastname: Fujisawa
authors.affiliation: Molecular Cytology Core Facility, Sloan Kettering Institute for Cancer Research, Memorial Sloan Kettering Cancer Center, 1275 York Avenue, Box # 333, New York, NY, 10065, USA.
authors.forename: Ning
authors.initials: N
authors.lastname: Fan
authors.affiliation: Molecular Cytology Core Facility, Sloan Kettering Institute for Cancer Research, Memorial Sloan Kettering Cancer Center, 1275 York Avenue, Box # 333, New York, NY, 10065, USA.
authors.forename: Matthew
authors.initials: M
authors.lastname: Brendel
authors.affiliation: Molecular Cytology Core Facility, Sloan Kettering Institute for Cancer Research, Memorial Sloan Kettering Cancer Center, 1275 York Avenue, Box # 333, New York, NY, 10065, USA. k-manova@ski.mskcc.org.
authors.forename: Katia
authors.initials: K
authors.lastname: Manova-Todorova
mesh_terms: D000818:Animals; D057205:Automation, Laboratory; D015415:Biomarkers; D005455:Fluorescent Antibody Technique; D007091:Image Processing, Computer-Assisted / Q000379:methods; D007150:Immunohistochemistry / Q000379:methods; D017403:In Situ Hybridization / Q000379:methods*; D007413:Intestinal Mucosa / Q000378:metabolism*; D008247:Lysosomes / Q000378:metabolism*; D051379:Mice; D015347:RNA Probes; D012333:RNA, Messenger / Q000235:genetics*; D043562:Receptors, G-Protein-Coupled / Q000235:genetics* / Q000378:metabolism; D012984:Software
chemical_list: D015415:Biomarkers; C493597:Lgr5 protein, mouse; D015347:RNA Probes; D012333:RNA, Messenger; D043562:Receptors, G-Protein-Coupled
keywords: Colorimetric and fluorescence mRNA detection; Immunofluorescence (IF); Intestinal crypt; Leica Bond RX; Mouse intestine; RNA Scope 2.5 LS; Stem cells; lysozyme; mLgr5 mRNA in situ hybridization