Detection of two different mRNAs in a single section by dual in situ hybridization: A comparison between colorimetric and fluorescent detection

Mapping a multiplexed zoo of mRNA expression

“…All N target mRNAs may be detected in parallel using N nucleic acid probe sets (each comprising one or more probes) that hybridize to orthogonal subsequences along the targets. If the background is sufficiently low, probes can be direct-labeled with reporter molecules to enable straightforward multiplexing (Kislauskis et al, 1993;Femino et al, 1998;Levsky et al, 2002;Kosman et al, 2004;Capodieci et al, 2005;Chan et al, 2005;Raj et al, 2008); in many settings, this approach does not yield sufficient contrast, so probes are instead used to mediate in situ signal amplification (Tautz and Pfeifle, 1989;Harland, 1991;Lehmann and Tautz, 1994;Kerstens et al, 1995;Nieto et al, 1996;Wiedorn et al, 1999;Player et al, 2001;Pernthaler et al, 2002;Thisse et al, 2004;Denkers et al, 2004;Kosman et al, 2004;Zhou et al, 2004;Larsson et al, 2004Larsson et al, , 2010Clay and Ramakrishnan, 2005;Barroso-Chinea et al, 2007;Acloque et al, 2008;Piette et al, 2008;Thisse and Thisse, 2008;Weiszmann et al, 2009;Wang et al, 2012).…”

Multidimensional quantitative analysis of mRNA expression within intact vertebrate embryos

“…Traditional in situ hybridization approaches based on catalytic reporter deposition (CARD) yield high-contrast images of mRNA expression domains within whole-mount vertebrate embryos (Tautz and Pfeifle, 1989;Harland, 1991;Lehmann and Tautz, 1994;Kerstens et al, 1995;Nieto et al, 1996;Pernthaler et al, 2002;Denkers et al, 2004;Kosman et al, 2004;Thisse et al, 2004;Clay and Ramakrishnan, 2005;Barroso-Chinea et al, 2007;Acloque et al, 2008;Piette et al, 2008;Thisse and Thisse, 2008;Weiszmann et al, 2009;RufZamojski et al, 2015). However, the intensity of the staining is qualitative rather than quantitative; furthermore, spatial resolution is often compromised by diffusion of reporter molecules prior to deposition (Tautz and Pfeifle, 1989;Thisse et al, 2004;Acloque et al, 2008;Piette et al, 2008;Thisse and Thisse, 2008;Weiszmann et al, 2009), and multiplexing is cumbersome, requiring serial staining of each target mRNA (Lehmann and Tautz, 1994;Nieto et al, 1996;Denkers et al, 2004;Kosman et al, 2004;Thisse et al, 2004;Clay and Ramakrishnan, 2005;Barroso-Chinea et al, 2007;Acloque et al, 2008;Piette et al, 2008).…”

“…However, the intensity of the staining is qualitative rather than quantitative; furthermore, spatial resolution is often compromised by diffusion of reporter molecules prior to deposition (Tautz and Pfeifle, 1989;Thisse et al, 2004;Acloque et al, 2008;Piette et al, 2008;Thisse and Thisse, 2008;Weiszmann et al, 2009), and multiplexing is cumbersome, requiring serial staining of each target mRNA (Lehmann and Tautz, 1994;Nieto et al, 1996;Denkers et al, 2004;Kosman et al, 2004;Thisse et al, 2004;Clay and Ramakrishnan, 2005;Barroso-Chinea et al, 2007;Acloque et al, 2008;Piette et al, 2008). These strengths and weaknesses all derive from the enzyme-mediated deposition process responsible for signal amplification.…”

Triple-Labeling Whole-Mount In Situ Hybridization Method for Analysis of Overlapping Gene Expression in Brain Tissue with High Level of Autofluorescence

“…Multicolor colorimetric in situ hybridization offers good sensitivity and is unaffected by autofluorescence. Many chromogenic multilabeling methods for detection of digoxigenin / biotin / fluoresceinlabeled probes have been developed [5][6][7]29,32]. Our strategy was first to combine the same alkaline phosphatase with Fast Red, INT/BCIP and NBT/BCIP substrates, which would provide reaction products of different colors.…”

“…With this technique, fluorescein, biotin and digoxigenin probes can be visualized using primary antibodies specific to the probe labels and secondary antibodies conjugated with brightly fluorescent dyes (Alexa or Cyanine). However, detection of low-abundance mRNA posed a challenge for conventional FISH, due to low sensitivity compared to chromogenic AP detection, because the latter can be amplified enzymatically [7]. Therefore, whole-mount FISH methods add a signal amplification step, using fluorescent tyramide substrates in sequential horseradish peroxidase reactions (tyramide signal amplification, TSA) [8][9][10].…”