Tsa plus kit

Manufactured by PerkinElmer

Sourced in United States

The TSA Plus kit is a laboratory instrument designed for the automated analysis of samples. It provides a standardized and efficient method for processing and analyzing various types of samples in a laboratory setting. The core function of the TSA Plus kit is to facilitate the processing and analysis of samples, enabling researchers and scientists to obtain accurate and reliable data.

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Lab products found in correlation

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18 protocols using tsa plus kit

Immunofluorescence Analysis of Intestinal Biopsies

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Cryo-frozen intestinal cross sections from patient colonic biopsies were fixed with 4% paraformaldehyde and stained using the TSA plus kit [Perkin Elmer] according to the manufacturer’s instructions. Alternatively, slides were fixed with methanol at −20°C for 10 min, blocked with 10% fetal calf serum [FCS]/1% bvine serum albumin for 1 h and incubated overnight at 4°C with the primary antibody. Paraffin-embedded tissues were deparaffinized and antigen unmasking was performed using citrate buffer. Thereafter slides were incubated with different antibodies.
For DSS-induced colitis mice, immunofluorescence analysis was performed in intestinal sections, which were taken at the end of the experiment on day 10 for IL10 [Abcam], IL17 [Abcam] and Foxp3 [eBioscience]. Colon biopsies of UC patients were taken before and 28 days after topical cobitolimod or placebo treatment^{21 (link),22 (link)} and the corresponding cross sections were stained for IL10 [Abcam], Foxp3 [eBioscience] or IL17 [Abcam]. From each sample, four to six high power fields [HPFs] per patient were analysed using a 10× objective magnification. Analysis of images was done with a fluorescence microscope [BZ-8100 or BZ-9000, Keyence] or a confocal microscope [LSM, Leica Microsystems] and calculated with ImageJ 1.52a [NIH]

Schmitt H., Ulmschneider J., Billmeier U., Vieth M., Scarozza P., Sonnewald S., Reid S., Atreya I., Rath T., Zundler S., Langheinrich M., Schüttler J., Hartmann A., Winkler T., Admyre C., Knittel T., Dieterich Johansson C., Zargari A., Neurath M.F, & Atreya R. (2019). The TLR9 Agonist Cobitolimod Induces IL10-Producing Wound Healing Macrophages and Regulatory T Cells in Ulcerative Colitis. Journal of Crohn's & Colitis, 14(4), 508-524.

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In Situ Hybridization Protocol for Marine Invertebrate Embryos

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RNA in situ hybridization was performed using Digoxigenin-11-UTP-labeled probes as previously described (McIntyre et al., 2013 (link)). Briefly, embryos were fixed in 4% paraformaldehyde overnight at 4°C, washed with ASW, and stored in methanol at −20°C. RNA probes were synthesized in vitro and used at 1 ng/μl. Hybridization took place at 60–65°C. Probes were visualized using alkaline phosphatase-conjugated anti-DIG antibody (1:1500, Roche [Indianapolis, IN, United States]). Finally, color was developed using NBT/BCIP (Roche). For double fluorescent in situ hybridization, a second probe (labeled with Fluorescein-12-UTP) was hybridized. Expression was visualized using the Tyramide Signal Amplification system (TSA-plus kit, Perkin Elmer [Waltham, MA, United States]). Embryos were visualized with a Zeiss Axioplan2 upright microscope.

Martik M.L, & McClay D.R. (2015). Deployment of a retinal determination gene network drives directed cell migration in the sea urchin embryo. eLife, 4, e08827.

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In-situ Hybridization of lncRNA KCNQ1OT1 and miR-34c-5p in Osteosarcoma

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A microarray containing tissue from 40 OS patients was obtained from Alena Biotechnology Co., Ltd. (Xi’an, China). OS tissue sections were hybridized with the lncRNA KCNQ1OT1 (BioTNT Biotechnologies, Shanghai, China) and miR-34c-5p probes (Servicebio, Wuhan, China). Probe mix was denatured at 85 °C and hybridization was conducted at 65 °C overnight. Sections were washed using reducing concentrations of saline sodium citrate. Then slides were treated with 5% blocking solution for 30 min at room temperature. Each section was incubated with 100 μl HRP-labeled anti-DIG antibody at 1:500 in blocking buffer overnight at 4 °C. Then washed with TBS and TSA staining solution was created with a Perkin-Elmer TSA Plus kit according to the manufacturer’s instructions. Incubated in DAPI-containing TBS, then rinsed in water, air dried, and mounted in an aqueous fluorescence mounting media. Confocal microscopy (LSM 510, META laser scanning microscope, Zeiss) was used to acquire the images. The intensities of KCNQ1OT1 and miR-34c-5p staining were scored using the following staining criteria: 0–5% was scored as 0; 6–35% was scored as 1; 36–70% was scored as 2; and >70% was scored as 3. A total score <2 was considered to represent negative expression, and a score ≥2 was defined as a positive expression. The scoring was performed blind and determined by two senior pathologists.

Shen Y., Xu J., Pan X., Zhang Y., Weng Y., Zhou D, & He S. (2020). LncRNA KCNQ1OT1 sponges miR-34c-5p to promote osteosarcoma growth via ALDOA enhanced aerobic glycolysis. Cell Death & Disease, 11(4), 278.

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Whole-Mount In Situ Hybridization Protocol

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Embryos were fixed in 4% paraformaldehyde in ASW for one hour at room temperature. Hybridization and chromogenic detection were performed using standard methods such as those described in (Walton et al., 2009 (link)), with the modification that 5% dextran sulfate was added to the hybridization buffer for increased sensitivity. DIG-labeled RNA probes were detected with an anti-DIG Fab AP antibody (Roche) and detected using NBT/BCIP. For double fluorescent detection, two probes, digoxigenin-11-UTP and fluorescein-12-UTP, were hybridized. Detection was carried out using cy3-tyramide and fluorescein tyramide reagents from the TSA-plus kit (Perkin Elmer). Hybridization and washing were performed at 65°C. Samples were imaged using a Zeiss Axioplan2 microscope. All staining patterns were quantified by scoring the expression patterns and each experiment was replicated a minimum of three times.

Warner J.F., Miranda E.L, & McClay D.R. (2016). Contribution of Hedgehog signaling to the establishment of left-right asymmetry in the sea urchin. Developmental biology, 411(2), 314-324.

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Immunohistochemical Analysis of Intestinal Tissues

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Cryo-frozen intestinal cross-sections were fixed with 4% paraformaldehyde (PFA) and stained using the TSA plus kit (Perkin Elmer, Baesweiler, Germany) according to the manufacturer’s instructions. Alternatively, slides were fixed with methanol at −20°C for 10 min, blocked with 10% fetal calf serum (FCS)/1% bovine serum albumin (BSA) for 1 hour and incubated overnight at 4°C with the primary antibody. Paraffin-embedded tissues were deparaffinised and antigen unmasking was performed using citrate buffer. After blocking, slides were incubated with TNFR2 (R&D, Minneapolis, Minnesota, USA), pSTAT3 (Cell Signalling, Leiden, Netherlands), CD3 (BD Biosciences, Heidelberg, Germany), Ki-67 (eBioscience, Frankfurt, Germany) or CD14 (Abcam, Cambridge, UK) antibodies. From each sample, 3–6 high power fields (HPF) per patient were analysed using ×10 objective magnification. For staining of active Caspase, the CaspACE FITC-VAD-FMK In Situ Marker (Promega, Mannheim, Germany) was used. TUNEL staining of PBMCs was performed with the TUNEL kit (Roche Diagnostics, Mannheim, Germany). Analysis of images was done with a fluorescence microscope (BZ-8100 or BZ-9000; Keyence, Neu-Isenburg, Germany) or a confocal microscope (LSM; Leica Microsystems, Wetzlar, Germany).

Schmitt H., Billmeier U., Dieterich W., Rath T., Sonnewald S., Reid S., Hirschmann S., Hildner K., Waldner M.J., Mudter J., Hartmann A., Grützmann R., Neufert C., Münster T., Neurath M.F, & Atreya R. (2018). Expansion of IL-23 receptor bearing TNFR2+ T cells is associated with molecular resistance to anti-TNF therapy in Crohn’s disease. Gut, 68(5), 814-828.

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Immunofluorescent Kidney Tissue Analysis

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For immunofluorescence, 15 μm cryostat sections of human kidney tissue from the fully anonymized archive at the Department of Pathology of the Medical University of Vienna ensuring compliance to the guidelines from the Medical University of Vienna and adherence to The Declaration of Helsinki (see section “Culture of primary HGMEC” above) were fixated and blocked. They were then incubated with 1:500 rabbit anti- NRGN polyclonal or anti-vWF monoclonal antibody for 1 hour. Primary antibodies were visualized using the TSA Plus Kit (PerkinElmer, Wellesley, MA, USA) and photographed with a fluorescence microscope.

Sengoelge G., Winnicki W., Kupczok A., von Haeseler A., Schuster M., Pfaller W., Jennings P., Weltermann A., Blake S, & Sunder-Plassmann G. (2014). A SAGE based approach to human glomerular endothelium: defining the transcriptome, finding a novel molecule and highlighting endothelial diversity. BMC Genomics, 15(1), 725.

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Immunohistochemical Analysis of Skin Samples

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Skin was fixed in 4% PFA for whole mount or in 10% formalin for paraffin embedding and used for histological analysis as previously described²⁶. Immunohistochemistry was performed by incubating sections at 4°C overnight with primary antibodies as follows: mouse anti-β-catenin (1:100, BD #610153; 14/Beta-Catenin), rat anti-CD11b (1:250, eBioscience #14-0112; M1/70), goat anti-P-cadherin (1:100, R&D #AF761), rabbit anti-pSmad2 (Ser465/467) (1:1000, Cell Signaling #3108; 138D4), and rabbit anti-Lef-1 (1:100, Cell Signaling #2286; C18A7). pSmad2 immunostaining required TSA Plus kit (PerkinElmer). For brightfield immunohistochemistry, biotinylated species-specific secondary antibodies, followed by detection using the ABC kit (Vector Labs) and DAB kit (Vector Labs), were used according to the manufacturer’s instructions. M.O.M. kit was used for mouse antibodies (Vector Laboratories). Secondary antibodies conjugated with FITC, RRX and Cy5 (Jackson Immunoresearch Laboratories) were used at a concentration of 1:100 for 1 hour at room temperature. Alexafluor 350 phalloidin (Life Technologies) was used according to the manufacturer’s instructions.

Mesa K.R., Rompolas P., Zito G., Myung P., Sun T.Y., Brown S., Gonzalez D., Blagoev K.B., Haberman A.M, & Greco V. (2015). Niche induced cell death and epithelial phagocytosis regulate hair follicle stem cell pool. Nature, 522(7554), 94-97.

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Immunofluorescence Staining of Zeb1 in Cells and Tissues

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IF analyses on cells and tissues were performed as described before (54 (link), 55 (link)) and outlined in SI Appendix, SI Materials and Methods. For Zeb1 staining on tissues an amplification step using the TSA Plus Kit (PerkinElmer) was performed according to manufactures instructions. Slides were mounted in ProLong Gold antifade reagent (Invitrogen).

Kröger C., Afeyan A., Mraz J., Eaton E.N., Reinhardt F., Khodor Y.L., Thiru P., Bierie B., Ye X., Burge C.B, & Weinberg R.A. (2019). Acquisition of a hybrid E/M state is essential for tumorigenicity of basal breast cancer cells. Proceedings of the National Academy of Sciences of the United States of America, 116(15), 7353-7362.

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Spinal Cord Immunohistochemistry in Arthritis Model

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At 15 and 54 days after anticollagen type-II antibody cocktail or saline injection, male and female mice were deeply anaesthetized and transcardially perfused with saline (0.9% NaCl) followed by a fixative solution (4% paraformaldehyde with 0.2% picric acid in 0.16 M phosphate buffer). Lumbar spinal cords were dissected, postfixed for 90 minutes at 4°C, and cryoprotected with 10% sucrose in 0.1 M phosphate buffer for 48 hours at 4°C. Spinal cords were then embedded in OCT compound (Tissue-Tek) and cut at 20 μm with a cryostat (Microm). Tissue sections were incubated overnight with primary antibodies against calcitonin gene–related peptide (CGRP) (1:32,000^{38 (link)}), substance P (SP) (1:4000^{13 (link)}), galanin (1:4000^{53 (link)}), glial fibrillary acidic protein (GFAP) (1:8000, Dako), and ionized calcium-binding adapter molecule 1 (IBA-1) (1:2000, Wako) at 4°C. Immunoreactivity was visualized with the TSA Plus kit (Perkin Elmer, Waltham, MA) as previously described.^{8 (link)} Images were captured with a LSM710 confocal laser scanning microscope (Carl Zeiss, Oberkochen, Germany), and the integrated signal intensity was measured after background subtraction in 3 sections per animal using Image J (NIH). Results are shown as the percentage change in signal intensity in CAIA compared with the saline group.

Fernandez-Zafra T., Gao T., Jurczak A., Sandor K., Hore Z., Agalave N.M., Su J., Estelius J., Lampa J., Hokfelt T., Wiesenfeld-Hallin Z., Xu X., Denk F, & Svensson C.I. (2018). Exploring the transcriptome of resident spinal microglia after collagen antibody–induced arthritis. Pain, 160(1), 224-236.

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Immunohistochemical Analysis of Skin Samples

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