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Tsa plus cy3 kit

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The TSA PLUS Cy3 kit is a reagent kit for signal amplification in immunohistochemistry (IHC) and in situ hybridization (ISH) applications. The kit provides a method for detecting and visualizing target proteins or nucleic acids in tissue sections or cell preparations using a horseradish peroxidase (HRP)-mediated fluorescent signal amplification system with Cy3 as the fluorescent label.

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Lab products found in correlation

Lsm 510 confocal laser scanning microscope, by Zeiss (2 mentions) 4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions) Fluoromount g, by Southern Biotech (1 mentions) Anti dig antibody, by Roche (1 mentions) Eclipse ti u inverted fluorescent microscope, by Nikon (1 mentions) Anti myc primary antibody 9b11, by Cell Signaling Technology (1 mentions) 4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions) Mouse anti cyclin d1, by Cell Signaling Technology (1 mentions) Streptavidin alexa 488, by Thermo Fisher Scientific (1 mentions) Pod conjugated anti dig antibodies, by Roche (1 mentions) Biotinylated anti ha antibodies, by BioLegend (1 mentions)

Spelling variants of this product

TSA kit (28 citations) TSA-Cy3 (21 citations) TSA Plus kit (18 citations) TSA Plus Cy3 (8 citations) TSA Cy3 kit (5 citations) Cy3-TSA plus amplification kit (4 citations)

5 protocols using tsa plus cy3 kit

1

Mapping GPCR Expression in Ai6;CRH-Cre Mice

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Sections from Ai6;CRH-Cre mice were hybridized with DIG-labeled GPCR riboprobes. After hybridization, sections were washed twice in 0.2× SSC at 63°C for 30 min and incubated at 37°C for 2 hours with peroxidase (POD)–conjugated anti-DIG antibodies (Roche, #11207733910; 1:2000) and goat anti-GFP antibodies to detect the Ai6 reporter ZsGreen (Rockland, #600-101-215; 1:1000). Sections were then washed three times for 5 min at room temperature (RT) in TNT [0.1 M tris-HCl (pH7.5), 0.5 M NaCl, and 0.05% Tween] buffer and then treated using the TSA Plus Cy3 Kit (PerkinElmer). Sections were then washed three times for 5 min at RT in TNT buffer and incubated with DAPI (4′,6-diamidino-2-phenylindole) (0.5 μg ml−1) and Alexa Fluor 488 donkey anti-goat immunoglobulin G (IgG) (Thermo Fisher Scientific, #A11055; 1:1000) at RT for 1 hour and washed. Sections were coverslipped with Fluoromount-G (Southern Biotech).
Lee E.J., Hanchate N.K., Kondoh K., Tong A.P., Kuang D., Spray A., Ye X, & Buck L.B. (2020). A psychological stressor conveyed by appetite-linked neurons. Science Advances, 6(12), eaay5366.
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2

Immunohistochemistry and RNAscope Analysis of Embryonic Expression

Cited 1 time
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Embryos were fixed overnight in 4% paraformaldehyde, then incubated
overnight in 10% followed by 30% sucrose at 4°C. 10μm transverse
cryosections were taken on Leica CM1900 crysostat (Leica Biosystems, Buffalo
Grove, IL).
Immunohistochemistry was performed on cryosections as previously
described with slight modifications for indirect antibody detection [19 (link)]. We used an anti-MYC primary antibody
(9B11, 1:1000; Cell Signaling #2276, Danvers, MA) followed by signal
amplification with goat anti-mouse IgG horseradish peroxidase (1: 500) (Perkin
Elmer Inc: NEF822001EA, Waltham, MA). The TSA plus Cy3 Kit (1:1500) (Perkin
Elmer Inc: NEL744001KT, Waltham, MA) was used for detection. The sections were
counterstained with 4′, 6-diamidino-2-phenylindole (DAPI, 1:10,000
dilution, Sigma-Aldrich). Images were obtained on a Nikon Eclipse Ti-U inverted
fluorescent microscope (Nikon Instruments, Melville, NY)
The sox11a RNAscope probe was obtained from Advanced
Cell Diagnostics (ACD: 590461, Newark, CA) and labelling was carried out on
cryosections according to RNAscope™ Multiplex Fluorescent V2
Assay protocol.
At least 10 embryos were analyzed per timepoint, and 3 separate
biological replicates were performed for each experiment.
Krueger L.A, & Morris A.C. (2022). Generation of a zebrafish knock-in line expressing MYC-tagged Sox11a using CRISPR/Cas9 genome editing. Biochemical and biophysical research communications, 608, 8-13.
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3

Immunohistochemical Analysis of Zebrafish Embryos

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Heat-shocked embryos were fixed using 4% paraformaldehyde overnight. For IHC, we used the following primary antibodies: rabbit anti-phopho-akt T308 (1:250, Cell Signaling Technology) and mouse anti-cyclin D1 (1:200, Cell Signaling Technology). For fluorescent detection of antibody labeling, we used Alexa Fluor 568 goat anti-rabbit, anti-mouse (1:500, Molecular Probes). Fluorescent in situ RNA hybridization was performed using TSA PLUS Cy3 kit (Perkin Elmer) as described previously (36 (link)). We used the following primers to generate probes for nanog: forward: 5′-ATGGCGGACTGGAAGATGCC-3′ reverse: 5′-ACAGCAAAGTTATTCCTTTAGTTGCC-3′. Fluorescence images were collected using a Zeiss LSM 510 laser scanning confocal microscope. We quantified the number of EGFP+Cy3+ cells throughout the whole zebrafish trunk. All data are displayed graphically as the mean ± SEM and comparisons of two groups were made using an unpaired Student t test. Mean values were considered statistically significant when P < 0.05.
Oh S.J., Cho H., Kim S., Noh K.H., Song K.H., Lee H.J., Woo S.R., Kim S., Choi C.H., Chung J.Y., Hewitt S.M., Kim J.H., Baek S., Lee K.M., Yee C., Park H.C, & Kim T.W. (2018). Targeting Cyclin D-CDK4/6 Sensitizes Immune-Refractory Cancer by Blocking the SCP3–NANOG Axis. Cancer research, 78(10), 2638-2653.
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4

Immunohistochemical Visualization of Proteins

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After hybridization, sections were washed twice in 0.2× SSC
at 63°C for 30 minutes, incubated with POD-conjugated anti-DIG
antibodies (Roche, #11207733910, 1:2000) and biotinylated anti-HA
antibodies (BioLegend, #901505, 1:300) at 37°C for 2 hours,
and then treated using the TSA-plus Cy3 kit (Perkin Elmer). Sections were
then incubated with 0.5 μg/ml DAPI and Alexa488 Streptavidin (Thermo
Fisher, 1:1000) at room temperature for 1 hour, and were coverslipped with
Fluoromount-G.
Kondoh K., Lu Z., Ye X., Olson D.P., Lowell B.B, & Buck L.B. (2016). A specific area of olfactory cortex involved in stress hormone responses to predator odors. Nature, 532(7597), 103-106.
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5

RNA Localization in Zebrafish Embryos

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To make an antisense probe, we designed a PCR primer (fgf2 F: 5′-CAGAGACCGACAGACTTAGGG-3′, fgf2 R: 5′-GCATTCCTCACAGTCAGACG-3′, nanog F: 5′-ATGGCGGACTGGAAGATGCC-3′, nanog R: 5′-ACAGCAAAGTTATTCCTTTAGTTGCC-3′, klf4 F: 5′-ATGGCTCTTGCAGATGCG-3′, klf4 R: 5′-ACATGTGCCTCTTCATGTGCAG-3′, sox2 F: 5′-ATGTATAACATGATGGAAACCGAGCTG-3′, sox2 R: 5′-CATATGCGATAAGGGAATCGTGCCG-3′, oct4 F: 5′-ATGACGGAGAGAGCGCAGAGC-3′, oct4 R: 5′-AGCTGGTGAGATGACCCACCAA-3′) and amplified a product from 1-day post-fertilization complementary DNA. The 24 hpf embryos were fixed using 4% paraformaldehyde overnight. Fluorescent in situ RNA hybridization was performed using TSA PLUS Cy3 kit (Perkin-Elmer, Wellesley, MA, USA) as previously described.32 Fluorescence images were collected using a Zeiss LSM 510 laser scanning confocal microscope (Carl Zeiss, Jena, Germany). We quantified the number of EGFP+Cy3+ cells throughout the whole zebrafish trunk. Statistical significance was tested with a nonpaired Student's t-test.
Song K.H., Cho H., Kim S., Lee H.J., Oh S.J., Woo S.R., Hong S.O., Jang H.S., Noh K.H., Choi C.H., Chung J.Y., Hewitt S.M., Kim J.H., Son M., Kim S.H., Lee B.I., Park H.C., Bae Y.K, & Kim T.W. (2017). API5 confers cancer stem cell-like properties through the FGF2-NANOG axis. Oncogenesis, 6(1), e285-.
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