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Signalstain ab diluent

Manufactured by Cell Signaling Technology
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SignalStain Ab Diluent is a ready-to-use buffer solution designed to dilute antibodies for immunohistochemistry and Western blotting applications. It is formulated to maintain antibody activity and specificity during the staining process.

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Lab products found in correlation

1.4 mm ceramic beads, by Qiagen (1 mentions) Citrate buffer, by Thermo Fisher Scientific (1 mentions) Tris edta buffer, by Abcam (1 mentions) Signalstain dab, by Cell Signaling Technology (1 mentions) Vectastain abc reagent, by Vector Laboratories (1 mentions) Superblock, by Thermo Fisher Scientific (1 mentions) Vector novared substrate, by Vector Laboratories (1 mentions) Fast green, by Electron Microscopy Sciences (1 mentions)

4 protocols using signalstain ab diluent

1

Biochemical Analysis of Akt/mTOR Pathway

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Antibodies to phospho‐Akt S473 (4060, 9271), phospho‐Akt T308 (9275), Akt (4691, 9272), Rictor (2140), phospho‐mTOR S2481 (2974), mTOR (2972), and phospho‐Stat3 Y705 (9145) were from Cell Signaling Technology. The β‐actin HRP (ab49900) antibody was from Abcam. Antibody to NeuN (MAB377) was from EMD Millipore Sigma. The Alexa Fluor 488 (A11029) and Alexa Fluor 594 (A11012) antibodies were purchased from Thermo Fisher Invitrogen. Vectashield Mounting Media with DAPI (H‐1200) was purchased from VWR. SignalStain Ab Diluent (8112) was purchased from Cell Signaling Technology. DAKO® Protein Block Serum‐Free was purchased from Fisher Scientific. Tough Tubes with Caps (13119‐500) and 1.4‐mM ceramic beads (13113‐325) were purchased form Mo‐Bio Laboratories. Protease and phosphatase inhibitor cocktail tablets were from Thermo Fisher.
Chellappa K., Brinkman J.A., Mukherjee S., Morrison M., Alotaibi M.I., Carbajal K.A., Alhadeff A.L., Perron I.J., Yao R., Purdy C.S., DeFelice D.M., Wakai M.H., Tomasiewicz J., Lin A., Meyer E., Peng Y., Arriola Apelo S.I., Puglielli L., Betley J.N., Paschos G.K., Baur J.A, & Lamming D.W. (2019). Hypothalamic mTORC2 is essential for metabolic health and longevity. Aging Cell, 18(5), e13014.
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2

Immunohistochemical Staining of Tissue Sections

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Deparaffinized 5μm-thick tissue sections were immunostained using the Vectastain R.T.U Elite ABC KIT according to manufacturer’s instructions. Briefly, antigen retrieval was performed by heating slides either in TRIS-EDTA buffer (pH 9) (Abcam) or with citrate buffer (pH 6) (Thermo Scientific) in a microwave and sections were sequentially blocked for 10 minutes with BLOXALL Blocking Solution (SP-6000), wash in PBS and blocked for 1hr with 10% horse blocking serum. Slides were then incubated for overnight in 4°C with primary antibody diluted in SignalStain Ab Diluent (Cell Signaling) Antibodies were used at the following concentrations: anti–pan-p63 (clone 4A4) 1/400, and anti-CK8/18 antibody (ab53280) 1/75. For all antibodies appropriate secondary Vectastain ABC Elite Kit HRP-conjugated antibodies and chromogenic 3,3-diaminobenzidine substrate were then successively added. Nuclei were counterstained with Hematoxylin. After staining, sections were dehydrated and mounted in the Permaslip medium (Alban Scientific Inc). Samples were visualized under a light microscope (Olympus BX50) and analyzed by Cell Sens Standard ver. 1.15 software.
Sandoval G.J., Pulice J.L., Pakula H., Schenone M.A., Takeda D.Y., Pop M., Boulay G., Williamson K.E., McBride M.J., Pan J., Pierre R.S., Hartman E., Garraway L.A., Carr S.A., Rivera M.N., Li Z., Ronco L., Hahn W.C, & Kadoch C. (2018). Binding of TMPRSS2-ERG to BAF chromatin remodeling complexes mediates prostate oncogenesis. Molecular cell, 71(4), 554-566.e7.
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3

Immunohistochemical Staining of Tissue Sections

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Tissue sections were de-paraffinized and re-hydrated. Antigen retrieval was performed in citrate buffer (pH 6.0) at 120°C for 10 min. Sections were treated with 3% H2O2 and blocked with normal goat serum in 0.05% TBS-T for 60 min and washed x3 with 0.05% TBS-T. Primary antibodies were diluted in SignalStain Ab Diluent (Cell Signaling Technology). Sections were incubated overnight at 4°C with primary antibody, washed 5 min in 0.05% TBS-T and then probed with SignalStain Boost Detection Reagent-rabbit or mouse (Cell Signaling Technology) for 30 min at room temperature in a humidified chamber. Visualization was carried out with SignalStain DAB (Cell Signaling Technology). Sections were counterstained with hematoxylin. Antibodies against the following antigens were used: HA-tag (CST-3724) 1:500; phospho-AKT (Ser 473) (CST-4060) 1:100; Ki67 (MKI67) (UMAB107)1:300.
Yoo J.H., Brady S.W., Acosta-Alvarez L., Rogers A., Peng J., Sorensen L.K., Wolff R.K., Mleynek T., Shin D., Rich C.P., Kircher D.A., Bild A., Odelberg S.J., Li D.Y., Holmen S.L, & Grossmann A.H. (2019). The small GTPase ARF6 activates PI3K in melanoma to induce a pro-metastatic state. Cancer research, 79(11), 2892-2908.
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4

PCNA Immunohistochemistry in Decalcified Bone

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In order to assess expression of proliferating cell nuclear antigen (PCNA) in vivo, decalcified, paraffin-embedded femur sections were subjected to immunohistochemical analysis, as previously described [38 (link)]. Briefly, slides were deparrafinized in xylenes, rehydrated in sequential alcohol baths, and incubated in 10 mM sodium citrate buffer for 10 minutes at 37°C and 30 minutes at room temperature to unmask antigens. Sections were blocked with SuperBlock (Thermo Scientific) for 1 hour and incubated overnight at 4°C in rabbit anti-PCNA antibody (1:1000) in SignalStain Ab Diluent (Cell Signaling Technology). The following day, slides were incubated with biotinylated secondary antibody and the Vectastain ABC reagent (Vector Laboratories) according to manufacturer’s instructions. Slides were stained with Vector NovaRED substrate (Vector Laboratories) and counterstained with Fast Green (Electron Microscopy Sciences). Slides were rapidly dehydrated through alcohols and xylenes prior to mounting with Permount and coverslips.
Buo A.M., Tomlinson R.E., Eidelman E.R., Chason M, & Stains J.P. (2017). Connexin43 and Runx2 Interact to Affect Cortical Bone Geometry, Skeletal Development, and Osteoblast and Osteoclast Function. Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research, 32(8), 1727-1738.
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