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Figure 5 | BMC Developmental Biology

Figure 5

From: In situ hybridization protocol for enhanced detection of gene expression in the planarian Schmidtea mediterranea

Figure 5

Improved signal intensity in regenerates and for low-abundance transcripts . (A-B) Single confocal sections of three day regenerating planarians labeled with DAPI (blue), the neoblast marker smedwi-1 (magenta), and the differentiating cell marker Smed-AGAT-1 (green). While gene expression is detectable in the intact tissue of planarians permeabilized with Proteinase K (A) or following HIAR (B), the superficial layers of the blastema (asterisk), including the AGAT-1-expressing cells, are lost following Proteinase K treatment. Dashed line denotes plane of amputation. (C-D) Maximum intensity projection of animals hybridized with DIG-labeled RNA probes for Smed-nog1 with a standard single TSA using TAMRA-conjugated tyramide (C) or iterative TSA first using DNP-tyramide followed by washing, incubation with peroxidase-conjugated anti-DNP antibody, and a second TSA reaction with TAMRA-conjugated tyramide (D). (C’-D”’) Maximum intensity projections of regions from the planarians in C and D, including the brain (C’ and D’), ventral nerve cord (C” and D”) and the lateral margin (C”’ and D”’). Scale bars: 100 μm.

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