Ttf 1

Manufactured by Santa Cruz Biotechnology

Sourced in United States

TTF-1 is a protein that functions as a transcription factor, playing a role in the regulation of gene expression. It is involved in the development and differentiation of various cell types, including those found in the thyroid, lung, and central nervous system. TTF-1 is often used as a marker in research and diagnostic applications.

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Anti-TTF-1 (3 citations)

7 protocols using ttf 1

Immunostaining of Cells and CTCs

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Cells in well plates were fixed with 4% PFA and permeabilized with 0.1% Triton in PBS. The cells were then blocked with 5% normal goat serum and 1% BSA in PBT solution. Cells were later immunostained with CK7/8 or TTF-1 (Santa Cruz Biotechnology) or Ki67 (Invitrogen) as well as the corresponding secondary antibodies. The number of CK7/8 positive cells was enumerated. After FACS sorting, CTCs were stained with EGFR (Cell Signaling) and pan-CK (Biolegend).

Zhang Z., Shiratsuchi H., Lin J., Chen G., Reddy R.M., Azizi E., Fouladdel S., Chang A.C., Lin L., Jiang H., Waghray M., Luker G., Simeone D.M., Wicha M.S., Beer D.G., Ramnath N, & Nagrath S. (2014). Expansion of CTCs from early stage lung cancer patients using a microfluidic co-culture model. Oncotarget, 5(23), 12383-12397.

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Western Blot Analysis of TTF-1 and GAPDH

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Protein samples were separated by SDS‐PAGE and subsequently blotted onto a polyvinylidene fluoride membrane. An iBind Western Device (Life Technologies Corporation, Carlsbad, CA, USA) was used for the antigen–antibody reaction. The membrane was incubated with antibodies against TTF‐1 (#sc‐53 136; Santa Cruz, Dallas, TX, USA) and GAPDH (#sc‐32 233; Santa Cruz). Bound antibodies were detected with horseradish peroxidase‐conjugated secondary mouse antibody (GE Healthcare Biosciences, Little Chalfont, UK), and images were taken using the Amersham Imager 680 (GE Healthcare Biosciences).

Sano K., Hayashi T., Suehara Y., Hosoya M., Takamochi K., Kohsaka S., Kishikawa S., Kishi M., Saito S., Takahashi F., Kaneko K., Suzuki K., Yao T., Ishijima M, & Saito T. (2021). Transcription start site‐level expression of thyroid transcription factor 1 isoforms in lung adenocarcinoma and its clinicopathological significance. The Journal of Pathology: Clinical Research, 7(4), 361-374.

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Immunohistochemical Analysis of Lung Tissues

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Lung tissues were harvested, fixed with 4% PFA in PBS and frozen, or paraffin embedded, sectioned and further processed. Tissue preparations were blocked with donkey or goat serum and incubated with anti-αSMA (Sigma-Aldrich, St. Louis, MO), SOX2, SOX9, TTF1, Endomucin, E-cadherin, HOPX (Santa Cruz, CA, USA), Pro-SPC and ADFP (Abcam, Cambridge, MA) followed by incubation with secondary antibodies conjugated to Alexa Fluor-488, 595 or 647 (Invitrogen, Carlsbad, CA) and counterstained with DAPI-containing mounting media. GFP signals were detected using a chicken polyclonal anti-GFP antibody (Abcam, Cambridge, MA). Data was analyzed by Imaris software, version 7.6.

Endale M., Ahlfeld S., Bao E., Chen X., Green J., Bess Z., Weirauch M.T., Xu Y, & Perl A.K. (2017). Temporal, spatial, and phenotypical changes of PDGFRα expressing fibroblasts during late lung development. Developmental biology, 425(2), 161-175.

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Immunohistochemical Staining Protocol

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Tissue sections or cell block were de-waxed in xylene and rehydrated in alcohol. Antigen retrieval was carried out by incubation in 10 mM citrate buffer (pH 6.0) at 95°C for 40 min. Endogenous peroxidase was blocked with 0.3% hydrogen peroxide for 10 min then incubated with 5% normal horse serum in phosphate-buffered saline (PBS) for 60 min at room temperature to block non-specific antibody reaction. After a wash with Tris-buffered saline plus 0.1% Tween 20(TBST), slides were incubated overnight at 4°C with primary antibodies, thyroid transcription factor-1 (TTF-1), Napsin A, and p-Hsp27 (sc-12359; 1:100, Santa Cruz Biotechnology, Inc., CA. USA). After being rinsed in TBST, slides were incubated for 30 min at room temperature with biotinylated secondary antibody followed by streptavidin–biotinylated–enzyme complex (streptABComplexes kit; Dako, Glostrup, Denmark). Subsequently, they were stained with 0.003% 3, 3-diaminobenzidine tetrahydrochloride, counterstained with Mayer's hematoxylin, dehydrated, and mounted.

Liu C.L., Chen S.F., Wu M.Z., Jao S.W., Lin Y.S., Yang C.Y., Lee T.Y., Wen L.W., Lan G.L, & Nieh S. (2016). The molecular and clinical verification of therapeutic resistance via the p38 MAPK–Hsp27 axis in lung cancer. Oncotarget, 7(12), 14279-14290.

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Immunofluorescence Staining of Adherent Cells

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Cells (1 × 10⁴) were plated into a well of an 8-well culture chamber slide (Falcon 354118) and incubated for 36 hours. Afterwards, cells were washed with PBS and blocked (0.3% Tritin X100, 1% BSA, 0.5% Normal Goat Serum in PBS). Primary antibodies were added for 75 minutes for β-catenin (Cell Signaling Technology, 8480S), TTF-1 (Santa Cruz Biotechnology, sc-13040), phalloidin-TRITC (Sigma Aldrich, P1951). Alexa-488 conjugated secondary goat-anti-rabbit antibody (Jackson ImmunoResearch, 111-545-045) was added after PBS wash for 60 min. After a final wash, mounting media with DAPI (Biotium, 23004) was added and a cover slip placed.

Phelps C.A., Lindsey-Boltz L., Sancar A, & Mu D. (2019). Mechanistic Study of TTF-1 Modulation of Cellular Sensitivity to Cisplatin. Scientific Reports, 9, 7990.

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Immunohistochemical Evaluation of Molecular Markers

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Immunohistochemical (IHC) staining was conducted using DAB substrate kit (ZSGB-BIO, Beijing, China) and the antibodies against Ki-67 (rabbit IgG, 1:150 dilution; Proteintech Group, Inc., Rosemont, IL, USA), PCNA, TTF-1 and Tg (Mouse monoclonal IgG, 1:50 dilution; Santa Cruz Biotech, Santa Cruz, CA, USA), NF-κB, COX-2 and IL-6 (Mouse monoclonal IgG, 1:50 dilution; Santa Cruz Biotech, Santa Cruz, CA, USA) and IkBα (1:100 from Cell Signaling, Cat., Danvers, MA, USA), respectively [26 (link)]. The results were evaluated according to the labeling intensity and scored as negative (−), weakly positive (+), moderately positive (++), and strongly positive (+++).

Zheng X., Jia B., Song X., Kong Q.Y., Wu M.L., Qiu Z.W., Li H, & Liu J. (2018). Preventive Potential of Resveratrol in Carcinogen-Induced Rat Thyroid Tumorigenesis. Nutrients, 10(3), 279.

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Histological and Immunohistochemical Characterization of NSCLC Patient-Derived Xenografts

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Each established xenograft model was bisected, fixed in 10% neutral buffered formalin and embedded in paraffin. Sections 5 um thick were cut from each paraffin‐embedded sample and mounted on glass slides. Routine hematoxylin and eosin staining of xenograft tumor sections was performed under an optical microscope. Tissue sections selected for immunohistochemistry (IHC) were routinely deparaffinized in xylene and rehydrated using a series of graded ethanol solutions, followed by antigen retrieval and background blocking. Primary antibodies against CD56, p63 (Cell Signaling Technology, Danvers, MA, USA), and thyroid transcription factor‐1 (TTF1; Santa Cruz Biotechnology Inc., Dallas, Texas, USA) were used following the manufacturer's instructions. The negative control was prepared following the same steps, except that the primary antibodies were replaced by normal serum from the same species. Two independent pathologists reviewed the tissue sections and compared pathologic similarities between NSCLC BDXs and corresponding patient samples.

Fu S., Zhao J., Bai H., Duan J., Wang Z., An T, & Wang J. (2015). High‐fidelity of non‐small cell lung cancer xenograft models derived from bronchoscopy‐guided biopsies. Thoracic Cancer, 7(1), 100-110.

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