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A method for combining RNAscope in situ hybridization with immunohistochemistry in thick free-floating brain sections and primary neuronal cultures.

Grabinski TM, Kneynsberg A, Manfredsson FP, Kanaan NM - PLoS ONE (2015)

Bottom Line: RNAscope ISH is a commercially available technology that utilizes a branched or "tree" in situ method to obtain ultrasensitive, single transcript detection.Immunohistochemistry is a tried and true method for identifying specific protein in cell populations.The combination of a sensitive and versatile oligonucleotide detection method with an established and versatile protein assay is a significant advancement in studies using free-floating tissue sections.

View Article: PubMed Central - PubMed

Affiliation: Department of Translational Science and Molecular Medicine, College of Human Medicine, Michigan State University, Grand Rapids, MI, United States of America.

ABSTRACT
In situ hybridization (ISH) is an extremely useful tool for localizing gene expression and changes in expression to specific cell populations in tissue samples across numerous research fields. Typically, a research group will put forth significant effort to design, generate, validate and then utilize in situ probes in thin or ultrathin paraffin embedded tissue sections. While combining ISH and IHC is an established technique, the combination of RNAscope ISH, a commercially available ISH assay with single transcript sensitivity, and IHC in thick free-floating tissue sections has not been described. Here, we provide a protocol that combines RNAscope ISH with IHC in thick free-floating tissue sections from the brain and allows simultaneous co-localization of genes and proteins in individual cells. This approach works well with a number of ISH probes (e.g. small proline-rich repeat 1a, βIII-tubulin, tau, and β-actin) and IHC antibody stains (e.g. tyrosine hydroxylase, βIII-tubulin, NeuN, and glial fibrillary acidic protein) in rat brain sections. In addition, we provide examples of combining ISH-IHC dual staining in primary neuron cultures and double-ISH labeling in thick free-floating tissue sections from the brain. Finally, we highlight the ability of RNAscope to detect ectopic DNA in neurons transduced with viral vectors. RNAscope ISH is a commercially available technology that utilizes a branched or "tree" in situ method to obtain ultrasensitive, single transcript detection. Immunohistochemistry is a tried and true method for identifying specific protein in cell populations. The combination of a sensitive and versatile oligonucleotide detection method with an established and versatile protein assay is a significant advancement in studies using free-floating tissue sections.

No MeSH data available.


Related in: MedlinePlus

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.
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pone.0120120.g004: 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.

Mentions: Semi-quantitative analysis of the ISH signal is possible in ISH/IHC stained tissue sections. The use of hue, saturation and brightness (HSB) color thresholding allows one to distinguish the two signals with a surprising degree of specificity, albeit not an absolutely specific method (i.e. some ISH puncta are not analyzed). Using ImageJ software, the thresholds were set to 0–128 for hue, 0–255 for saturation and 0–141 for brightness to distinguish the DAB ISH signal from the Vector SG IHC signal. This method was used to successfully measure the level of ISH signal in regional analyses using the entire image area (Fig. 4). Analyses of the pixels identified by the threshold settings (number of objects, area, fraction of the total area and size of objects) confirm the significant increase of Sprr1a in the lesioned hemisphere compared to the intact hemisphere (Fig. 4C-4F). Moreover, these settings allowed us to distinguish 4 levels of ISH signal as demonstrated by measurements made in individual cells with varying degrees of ISH signal (Fig. 5A-5D). Here, the same thresholding parameters were applied to individual neurons. Again, number of objects, area, fraction of the total area and size of objects can identify different populations of neurons with varying amounts of ISH signal (Fig. 5E-5H). Ultimately, the settings for thresholding need to be determined empirically with each stain. The goal for quantitation should be to include as much ISH signal, while excluding as much IHC signal as possible. Collectively, these data demonstrate that ISH/IHC in thick free-floating brain sections can be analyzed using semi-quantitative analyses.


A method for combining RNAscope in situ hybridization with immunohistochemistry in thick free-floating brain sections and primary neuronal cultures.

Grabinski TM, Kneynsberg A, Manfredsson FP, Kanaan NM - PLoS ONE (2015)

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.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4368734&req=5

pone.0120120.g004: 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.
Mentions: Semi-quantitative analysis of the ISH signal is possible in ISH/IHC stained tissue sections. The use of hue, saturation and brightness (HSB) color thresholding allows one to distinguish the two signals with a surprising degree of specificity, albeit not an absolutely specific method (i.e. some ISH puncta are not analyzed). Using ImageJ software, the thresholds were set to 0–128 for hue, 0–255 for saturation and 0–141 for brightness to distinguish the DAB ISH signal from the Vector SG IHC signal. This method was used to successfully measure the level of ISH signal in regional analyses using the entire image area (Fig. 4). Analyses of the pixels identified by the threshold settings (number of objects, area, fraction of the total area and size of objects) confirm the significant increase of Sprr1a in the lesioned hemisphere compared to the intact hemisphere (Fig. 4C-4F). Moreover, these settings allowed us to distinguish 4 levels of ISH signal as demonstrated by measurements made in individual cells with varying degrees of ISH signal (Fig. 5A-5D). Here, the same thresholding parameters were applied to individual neurons. Again, number of objects, area, fraction of the total area and size of objects can identify different populations of neurons with varying amounts of ISH signal (Fig. 5E-5H). Ultimately, the settings for thresholding need to be determined empirically with each stain. The goal for quantitation should be to include as much ISH signal, while excluding as much IHC signal as possible. Collectively, these data demonstrate that ISH/IHC in thick free-floating brain sections can be analyzed using semi-quantitative analyses.

Bottom Line: RNAscope ISH is a commercially available technology that utilizes a branched or "tree" in situ method to obtain ultrasensitive, single transcript detection.Immunohistochemistry is a tried and true method for identifying specific protein in cell populations.The combination of a sensitive and versatile oligonucleotide detection method with an established and versatile protein assay is a significant advancement in studies using free-floating tissue sections.

View Article: PubMed Central - PubMed

Affiliation: Department of Translational Science and Molecular Medicine, College of Human Medicine, Michigan State University, Grand Rapids, MI, United States of America.

ABSTRACT
In situ hybridization (ISH) is an extremely useful tool for localizing gene expression and changes in expression to specific cell populations in tissue samples across numerous research fields. Typically, a research group will put forth significant effort to design, generate, validate and then utilize in situ probes in thin or ultrathin paraffin embedded tissue sections. While combining ISH and IHC is an established technique, the combination of RNAscope ISH, a commercially available ISH assay with single transcript sensitivity, and IHC in thick free-floating tissue sections has not been described. Here, we provide a protocol that combines RNAscope ISH with IHC in thick free-floating tissue sections from the brain and allows simultaneous co-localization of genes and proteins in individual cells. This approach works well with a number of ISH probes (e.g. small proline-rich repeat 1a, βIII-tubulin, tau, and β-actin) and IHC antibody stains (e.g. tyrosine hydroxylase, βIII-tubulin, NeuN, and glial fibrillary acidic protein) in rat brain sections. In addition, we provide examples of combining ISH-IHC dual staining in primary neuron cultures and double-ISH labeling in thick free-floating tissue sections from the brain. Finally, we highlight the ability of RNAscope to detect ectopic DNA in neurons transduced with viral vectors. RNAscope ISH is a commercially available technology that utilizes a branched or "tree" in situ method to obtain ultrasensitive, single transcript detection. Immunohistochemistry is a tried and true method for identifying specific protein in cell populations. The combination of a sensitive and versatile oligonucleotide detection method with an established and versatile protein assay is a significant advancement in studies using free-floating tissue sections.

No MeSH data available.


Related in: MedlinePlus