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Tyramide superboost kit

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The Tyramide SuperBoost kit is a reagent system designed to amplify signals in immunohistochemistry (IHC) and in situ hybridization (ISH) experiments. The kit utilizes a proprietary tyramide-based signal amplification technology to enhance the detection of target antigens or nucleic acid sequences.

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Lab products found in correlation

Bromodeoxyuridine (brdu), by Abcam (3 mentions) Axio observer z1, by Zeiss (3 mentions) Goat anti rabbit igg hrp conjugate, by Thermo Fisher Scientific (3 mentions) Goat anti mouse igg hrp conjugate, by Thermo Fisher Scientific (3 mentions) Mouse anti brdu, by BD (3 mentions) Alexa fluor secondary antibody, by Thermo Fisher Scientific (2 mentions) Primary antibodies against pdgfrα, by Thermo Fisher Scientific (2 mentions) Hla dr tal1b5, by Santa Cruz Biotechnology (2 mentions) Panck ae1 ae3, by Biocare Medical (2 mentions) Hla dr dp dq dx sc 53302, by Biocare Medical (2 mentions) Sp5 2 sted cw confocal microscope, by Leica camera (2 mentions) Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions) Rabbit anti phospho histone3 ser10, by Merck Group (2 mentions) Fluorescein conjugated lectin concanavalin a, by Vector Laboratories (2 mentions) Taz wwtr1, by Cell Signaling Technology (2 mentions) Tead1, by Abcam (2 mentions) Lsm 700 confocal microscope, by Zeiss (2 mentions) Neutral buffered formalin, by Thermo Fisher Scientific (1 mentions) Antigen retrieval citra plus, by BioGenex (1 mentions) 3 3 diaminobenzidine (dab), by Vector Laboratories (1 mentions)

39 protocols using tyramide superboost kit

1

Detailed Immunofluorescence Staining Protocol

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Deparaffinisation and antigen retrieval was carried out using the PT-Link System (DAKO), followed by blocking in 10% of either donkey or goat serum. Tissue sections were incubated with primary antibodies at 4 °C overnight followed by incubation with fluorophore-conjugated secondary antibodies or tyramide signal amplification using Tyramide Superboost Kit (Thermo Fisher) and nuclear dye DAPI (Thermo Fisher) at room temperature for 2 h. Antibodies used are listed in Supplementary Table 1. Tissue sections were mounted using Fluorescent Mounting Media (DAKO). For staining using two antibodies raised from the same host species, tyramide signal amplification was performed using Tyramide Superboost Kit (Thermo Fisher) according to manufacturer’s guidelines. For pSTAT3 (CST#9145, 1:100) staining, fresh frozen OCT embedded tissue was sectioned and pre-treated with methanol for 10 min at −20 °C before proceeding with immunofluorescence staining. Tissue sections were imaged using an Axio Observe Light Microscope with the Apotome.2 (Zeiss) and quantified using ImageJ and Zen Black software. From each mouse or patient, a minimum of three fields of view, per section, were quantified and averaged for each data point.
Raymant M., Astuti Y., Alvaro-Espinosa L., Green D., Quaranta V., Bellomo G., Glenn M., Chandran-Gorner V., Palmer D.H., Halloran C., Ghaneh P., Henderson N.C., Morton J.P., Valiente M., Mielgo A, & Schmid M.C. (2024). Macrophage-fibroblast JAK/STAT dependent crosstalk promotes liver metastatic outgrowth in pancreatic cancer. Nature Communications, 15, 3593.
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2

Immunohistochemical and Immunofluorescent Staining of Pancreatic Tissues

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Pancreatic tissues were fixed in 10% neutral-buffered formalin (FisherBrand) and then embedded in paraffin and sectioned into slides. For IHC, fresh-cut paraffin slides were rehydrated using two series of xylene, two series of 100% ethanol, and then two series of 95% ethanol. Water was used to wash all residues from previous washes. Antigen retrieval was performed using Antigen Retrieval CITRA Plus (BioGenex) and microwaved for a total of 8 min. Upon cool-down, tissue was blocked using 1% BSA in PBS for 30 min, and then primary antibodies in Table S1 were used at their corresponding dilutions. Biotinylated secondary antibodies were used in 1:300 dilution. Following the secondary antibody incubation, the tissue was incubated for 30 min with the ABC reagent from VECTASTAIN Elite ABC kit, peroxidase (Vector Laboratories). Then it was developed using DAB (Vector). For IF, Alexa fluor secondary antibodies (1:300, Invitrogen) were used. Prolong Diamond Antifade Mountant with DAPI (Invitrogen) was used for nuclei staining. The Tyramide SuperBoost Kit (Invitrogen) was used in IF when primary antibodies raised in the same species were used. Images were taken with an Olympus BX-53 microscope, Olympus DP80 digital camera, and Olympus cellSens standard software. Some IF images were acquired using the Leica Stellaris 5 confocal microscope (Leica Microsystems).
Du W., Menjivar R.E., Donahue K.L., Kadiyala P., Velez-Delgado A., Brown K.L., Watkoske H.R., He X., Carpenter E.S., Angeles C.V., Zhang Y, & Pasca di Magliano M. (2022). WNT signaling in the tumor microenvironment promotes immunosuppression in murine pancreatic cancer. The Journal of Experimental Medicine, 220(1), e20220503.
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3

Embryonic Mouse Brain Histology Protocol

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Embryonic mouse brains were dissected in PBS and fixed overnight in 4% paraformaldehyde (PFA) at 4°C. Postnatal animals were perfused with 0.9% saline and PFA, and their brains were dissected and fixed in PFA overnight at 4°C. Histology, immunostaining, and TUNEL assay were performed as described previously (Lavado et al., 2018 (link)). Luxol blue and cresyl violet staining was performed using the Kluver-Barrera method (EMS #26681). The primary antibodies used were: YAP/TAZ (Cell Signaling #8418, 1:500), YAP (Cell Signaling #14074, 1:250), TAZ (Cell Signaling #83669, 1:100 with the Tyramide SuperBoost Kit, Invitrogen #B40922), SOX2 (Santa Cruz Biotechnology #SC17320, 1:100), SOX2–Alexa-647 (BD Biosciences #562139, 1:100), TBR2 (Thermo #14-4875-82, 1:250), TBR2 (Abcam ab23345, 1:250), TBR1 (Abcam ab31940, 1:1000), CUX1 (Santa Cruz #SC13024, 1:250), CTIP2 (Abcam ab18465, 1:1000), PAX6 (BioLegend #901301, 1:500), BrdU/IdU (BD Biosciences #347580, 1:50), BrdU (Abcam ab152095, 1:500), Ki67 (Vector laboratories #VP-RM04, 1:500), ZO1–Alexa-488 (Thermo #339188, 1:100), Nestin (R&D systems #AF2736, 1:100), ARL13b (ProteinTech #17711-1-AP, 1:250), β-Catenin–Alexa-647 (Cell Signaling #4627, 1:100), and S100β (Sigma #52532, 1:100).
Lavado A., Gangwar R., Paré J., Wan S., Fan Y, & Cao X. (2021). YAP/TAZ Maintain the Proliferative Capacity and Structural Organization of Radial Glial Cells during Brain Development. Developmental biology, 480, 39-49.
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4

Immunofluorescence Staining of PDGFRα

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For the immunofluorescence staining of PDGFRα (Figure 3H), slides were blocked with 3% H2O2 for 15 minutes following fixation to inhibit endogenous peroxidases. Sections were the blocked for 1 hour with blocking buffer at RT. Tissues were incubated with primary antibodies against PDGFRα (1:300, eBioscience) for 3 hours, washed with 1X PBS, and stained with secondary antibodies for 3 hours (1:200) A Tyramide SuperBoost kit (Invitrogen) was used for the detection of PDGFRα per manufacturer instructions. For PDGFRα staining in Figure 4A, sections were stained with antibodies against PDGFRα (1:200, R and D systems) diluted in blocking buffer for 3 hours at RT, followed by staining with corresponding secondary antibody for 1 hour at RT.
Kastenschmidt J.M., Coulis G., Farahat P.K., Pham P., Rios R., Cristal T.T., Mannaa A.H., Ayer R.E., Yahia R., Deshpande A.A., Hughes B.S., Savage A.K., Giesige C.R., Harper S.Q., Locksley R.M., Mozaffar T, & Villalta S.A. (2021). A stromal progenitor and ILC2 niche promotes muscle eosinophilia and fibrosis-associated gene expression. Cell reports, 35(2), 108997.
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5

Multicolor Immunohistochemistry of FFPE Tissue

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Formalin-fixed paraffin-embedded (FFPE) tissue sections were cut at 4 μm and deparaffinized. Antigen retrieval was performed with citrate buffer pH 6. Endogen peroxidase were blocked and protein block was applied. Sections were then incubated with the primary antibody (HLA-DR TAL1B5 Santa Cruz at 1:4000, panCK AE1/AE3 Biocare at 1:400, HLA-DR/DP/DQ/DX sc-53302 at 1:2000) overnight at 4°C. Sections were then incubated with the secondary antibody and TSA reagent (Tyramide Superboost Kit cat#B40912, Invitrogen) applied according to manufacturer’s recommendations. The procedure was repeated one or two more times with the subsequent different primary antibodies and then counterstained with DAPI for nuclei identification. Tonsil was used as a positive control. A detailed protocol can be found in supplementary data.
Gonzalez-Ericsson P.I., Wulfkhule J.D., Gallagher R.I., Sun X., Axelrod M.L., Sheng Q., Luo N., Gomez H., Sanchez V., Sanders M., Pusztai L., Petricoin E., Blenman K.R, & Balko J.M. (2021). Tumor-Specific Major Histocompatibility-II Expression Predicts Benefit to Anti–PD-1/L1 Therapy in Patients With HER2-Negative Primary Breast Cancer. Clinical Cancer Research, 27(19), 5299-5306.
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6

Immunohistochemistry and Immunofluorescence Protocol

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Tissue preparation, immunohistochemistry and immunofluorescence were performed as previously described in [23 (link)]. For peroxidase Lyve-1 staining of 2-month-old mice and peroxidase podoplanin staining, tyramide-enhanced avidin-biotin immunostaining method was used (Tyramide SuperBoost™ Kit, Invitrogen, Carlsbad, USA; Vectatstain ABC Kit, Vector Laboratories, Eching, Germany).
Lohrberg M., Pabst R, & Wilting J. (2018). Co-localization of lymphoid aggregates and lymphatic networks in nose- (NALT) and lacrimal duct-associated lymphoid tissue (LDALT) of mice. BMC Immunology, 19, 5.
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7

Dystrophin Expression in Low-Grade Glioma

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Haematoxylin and eosin (H&E) and unstained formalin-fixed paraffin-embedded (FFPE) sections from 24 LGG cases were obtained from University Hospital Southampton NHS Foundation Trust as part of BRAIN UK and under the extended ethical approval of the South Central—Hampshire B Research Ethics Committee (REC reference: 19/SC/027, IRAS project ID: 262890). The cohort included 18 cases of astrocytoma, one case of oligodendroglioma and five cases of ‘not otherwise specified’. Nine out of the 24 cases were IDH mutant and 15 were IDH wild-type as previously determined by the providing centre. Sections were deparaffinised with xylene and rehydrated through graded alcohol. Antigen retrieval was achieved by immersing sections in pH 6.0 citrate buffer and heating in a 800 W microwave for 10 min at high power and 10 min at low power. Immunohistochemistry was performed using a C-terminal anti-dystrophin antibody (Abcam 15277) at a 1:100 dilution followed by a Tyramide SuperBoost™ kit (Invitrogen) according to manufactures’ instructions. Staining was visualised using 3,3′-diaminobenzidine tetra hydrochloride (DAB), and counterstained with haematoxylin.
Naidoo M., Jones L., Conboy B., Hamarneh W., D’Souza D., Anthony K, & Machado L.R. (2022). Duchenne muscular dystrophy gene expression is an independent prognostic marker for IDH mutant low-grade glioma. Scientific Reports, 12, 3200.
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8

Immunofluorescent Staining of TLR Receptors

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HFFs were plated at 104 per well in a tissue culture-treated 96-well plate and incubated overnight. Each well was washed 3 times in PBS and then fixed for 10 min at room temperature in 4% paraformaldehyde followed by blocking with 10% goat serum in PBS (recommended blocking buffer for the Tyramide SuperBoost kit described below) for 1 h at room temperature. Polyclonal anti-TLR1, −2, and −6 antibodies (Abcam, Cambridge, MA, Catalog numbers AB189337, AB191458, AB37072, respectively) were utilized at 1:100 dilutions in 100 µL blocking buffer to stain cells for 1 h at room temperature, followed by 1 h of incubation with goat-anti-rabbit-HRP antibody conjugate. Tyramide signal amplification was carried out for 5 min according to the Tyramide SuperBoost kit's manufacturer (Invitrogen, Carlsbad, CA, Catalog number B40922). Samples were counterstained with 100 ng of DAPI and imaged at the University of Minnesota Imaging Center using a Nikon A1R confocal microscope with a 60X water immersion objective with a numerical aperture of 1.20.
Salyer A.C, & David S.A. (2018). Transcriptomal signatures of vaccine adjuvants and accessory immunostimulation of sentinel cells by toll-like receptor 2/6 agonists. Human Vaccines & Immunotherapeutics, 14(7), 1686-1696.
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9

Immunohistochemical Staining of Drosophila Brains

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The protocol was adapted from the Flylight protocol (62 (link)). Briefly, brains were dissected, fixed, and stained with rabbit anti-hPNPO and mouse anti-Brp antibodies. Signals for hPNPO were amplified with the Tyramide SuperBoost kit (Invitrogen; catalog no. B40926). DAPI was added into the wash buffer to stain the nucleus when needed. Images were taken using a Leica SP5-II-STED-CW confocal microscope and processed in Fiji (63 (link)) (SI Appendix).
Chi W., Iyengar A.S., Fu W., Liu W., Berg A.E., Wu C.F, & Zhuang X. (2022). Drosophila carrying epilepsy-associated variants in the vitamin B6 metabolism gene PNPO display allele- and diet-dependent phenotypes. Proceedings of the National Academy of Sciences of the United States of America, 119(9), e2115524119.
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10

Immunofluorescence Protocol with Tyramide SuperBoost

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Immunofluorescence was performed according to an adapted protocol from Dakin S et al. with the incorporation of the Tyramide SuperBoost Kit (Invitrogen, Waltham, USA) (29 (link)). For detailed information, see the online Supplementary Material and Methods and Supplementary Table S1.
Hammitzsch A., Ossadnik A., Bachmann Q., Merwald-Fraenk H., Lorenz G., Witt M., Wiesent F., Mühlhofer H., Simone D., Bowness P., Heemann U., Arbogast M., Moog P, & Schmaderer C. (2024). Increased interleukin-26 in the peripheral joints of patients with axial spondyloarthritis and psoriatic arthritis, co-localizing with CD68-positive synoviocytes. Frontiers in Immunology, 15, 1355824.
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