Fig 1.
Illustration of RNAscope branched ISH method.
RNAscope utilizes a branched ISH method that provides significant amplification and discrete detection of single transcripts. (A) RNAscope ISH produces puncta of signal that represent a single mRNA transcript. It is noteworthy, that at a certain level the amount of transcript can be elevated to a level where individual puncta are difficult to discern within single cells (see Figs. 2I and 5D as examples). (B) This level of sensitivity is achieved by initially hybridizing multiple tandem “Z” probes to the target transcript (these primers typically target 50bp long sequences and multiple sets of primers are made against the target molecule, ACD offers custom designed primers). Then a pre-amplifier probe is bound to the tandem Z probe sets, followed by the addition of multiple amplifier probes to the pre-amplifier probe. Finally, a number of detection probes that are conjugated to horseradish peroxidase (HRP) enzyme are added onto the amplifier probes and HRP substrate is added (i.e. diaminobenzidine, DAB). The resulting conversion of DAB to a brown colored reaction product by all of the HRPs bound the branched ISH complex produces discrete puncta. Alternatively, alkaline phosphatase (two-plex RNAscope) or fluorophores (multi-plex RNAscope) are conjugated to the detection probes for the appropriate version of the assay.
Fig 2.
RNAscope ISH combined with IHC in thick free-floating rat brain tissue sections.
The dopaminergic nigrostriatal system of rats was unilaterally lesioned using 6-OHDA (a dopaminergic neurotoxin) delivered to the striatum. One week later the brains were collected and tissue was processed for Sprr1a ISH (brown) and tyrosine hydroxylase (TH) IHC (blue). A) Low magnification images clearly depict the loss of dopaminergic (i.e. TH+) neurons in the lesioned (right side) substantia nigra compared to the unlesioned hemisphere (left side). Note the presence of substantial Sprr1a ISH signal in the lesioned hemisphere, and the lack of signal in the unlesioned hemisphere. B and C) High magnification images of a 20μm thick section show clear dual labeling with ISH and IHC in the lesioned side (C), but not the unlesioned side (B). D and E) Images from a 40μm thick section show similar results. F-H) Neurons in the lesioned hemisphere exhibited little to no ISH with strong IHC (F), intermediate ISH and IHC (G), and strong ISH with little to no IHC (H). Scale bars: A = 500μm, B-E = 40μm and F-H = 20μm.
Fig 3.
Control sections for ISH-IHC dual labeling in thick free-floating sections.
A) Tissues were labeled with a rat positive control probe (i.e. rat peptidylprolyl isomerase B gene) provided by ACD. Clear ISH signal and IHC staining were observed. This probe acts as a positive control because it is present ubiquitously throughout rat tissue, albeit at modest levels in the cells depicted. B) Tissues were labeled with a negative control probe (i.e. Bacillus subtilis dihydrodipicolinate reductase gene) provided by ACD. Very infrequent ISH puncta are present, while strong IHC is clear. This probe was used as a negative control because it is a bacterial transcript not present in rat tissue. C) Tissue was labeled with Sprr1a ISH, but the primary TH antibody was excluded. As expected, strong Sprr1a ISH is apparent in the SN of the lesioned hemisphere (i.e. brown ISH puncta), but no TH IHC is present (i.e. lack of blue staining). D) To ensure the signals were dependent upon the Sprr1a ISH probe and TH primary antibody we omitted the Sprr1a probe and the TH primary antibody. As expected, this results in no signal for ISH (i.e. no brown puncta) or IHC (i.e. no blue staining). Scale bar: A-D = 50μm.
Fig 4.
Semi-quantitative regional analysis of ISH signal in dual labeled (ISH-IHC) thick tissue sections.
A-D) Images were processed using ImageJ software to set the HSB index and hue was 0–128, brightness was 0–255 and saturation was 0–141. The pixels within the threshold limit are indicated by the bright red overlay. These setting allowed a distinction with fair accuracy between the Sprr1a ISH signal (brown) and the TH IHC signal (blue). The red areas in C and D are the pixels analyzed after the thresholds were set. E-H) After applying the threshold settings, image analysis was used to measure the average number of objects (E), mean area of pixels (F), mean fraction of the total area (G), and mean size of objects (H) (*p<0.05 compared to intact). With each measurement, the lesioned side is significantly greater than the unlesioned hemisphere. Scale bar: A-D = 40μm.
Fig 5.
Semi-quantitative single-cell analysis of ISH signal in dual labeled (ISH-IHC) thick tissue sections.
A-D) Four groups of TH+ neurons were found with varying amounts of ISH signal in the lesioned hemisphere (using same threshold settings as in Fig. 3). Individual neurons contained no/very little (A and E), low (B and F), moderate (C and G), and high (D and H) levels of ISH signal. The red areas in E-H are the pixels analyzed after the thresholds were set. I-L) These groups were distinguishable using the mean object number (I), mean area (J), size of the objects (K) and the fraction of the total area (L). The object number measurement detected significant differences between all four groups (p<0.05), while all other measurements identified differences between the low, moderate and high level of ISH groups. Scale bar: A-H = 20μm.
Fig 6.
The versatility of ISH-IHC dual staining method.
A number of combinations with different ISH and IHC stains were performed. A) Rat βIII-tubulin ISH (brown) and βIII-tubulin IHC (blue) were combined to label the mRNA and protein of the same target. B) Rat βIII-tubulin ISH was combined with IHC for NeuN, a common neuron-specific marker. C) Rat β-actin ISH was combined with IHC for GFAP, a common astrocyte-specific marker. Both B and C confirm that cell type-specific markers are compatible with this ISH-IHC dual staining method. D) Sprr1a ISH was combined with GFAP IHC to demonstrate that the same ISH marker can be combined with multiple IHC markers (i.e. TH in Figs. 2–5 and GFAP here). All images were taken in the area of the retrosplenial cortex. Scale bar: A-D = 50μm.
Fig 7.
RNAscope ISH-IHC in primary neuron cultures, dual RNAscope ISH detection, and detection of ectopic viral vector DNA.
A) Primary rat neurons were processed for tau ISH and βIII-tubulin IHC. Clear puncta are present for the tau ISH signal (red), while neuronal cell bodies and processes are clearly labeled with tubulin IHC (blue). B-E) Color thresholding (using the YUV index) allows clear separation of ISH signal (red puncta) from the IHC signal (blue staining) in primary neurons. Here, the YUV index was used and Y was 0–135, U was 0–255 and V was 135–255. The pixels within the threshold limits are shown by the bright red overlay. F) Dual ISH for tau (red) and βIII-tubulin (blue/green) was performed on 40μm thick rat brain sections (cortex depicted). G-J) Rats were unilaterally injected in the striatum with recombinant adeno-associated viruses (rAAV, serotype 2/5). RNAscope was used to detect rAAV DNA. Robust rAAV DNA ISH signal is detected in the injected striatum (G) compared to the uninjected hemisphere (I). ISH signal was also detected in the SN ipsilateral to the injected striatum because rAAV particles are retrogradely transported to the SN (H), no signal is detected in the contralateral SN (J). Scale bars: A = 50μm, C and E = 20μm, F = 25μm, and J = 50μm.