Ab58655

Manufactured by Abcam

Sourced in United Kingdom, United States

Ab58655 is a primary antibody that targets the protein of interest. It is a polyclonal antibody produced in rabbit. The product is suitable for use in various immunoassay applications, including Western blot, immunohistochemistry, and ELISA.

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Lab products found in correlation

Ab34710, by Abcam (4 mentions) Ab203676, by Abcam (2 mentions) Ab7778, by Affinity Biosciences (2 mentions) Df6543, by Abcam (2 mentions) Ab108930, by Abcam (2 mentions) Ab92486, by Abcam (2 mentions) Triton x 100, by Thermo Fisher Scientific (2 mentions) Fluorescence microscope, by Olympus (1 mentions) Normal horse serum, by Vector Laboratories (1 mentions) Formalin, by Bio-Optica (1 mentions) 4 6 diamidino 2 phenylindole (dapi), by Biotium (1 mentions) A21206, by Thermo Fisher Scientific (1 mentions) Tcs sp8, by Leica camera (1 mentions) Phalloidin, by Merck Group (1 mentions) A1352, by ABclonal (1 mentions) A3795, by ABclonal (1 mentions) Ab58632, by Abcam (1 mentions) Ab150083, by Abcam (1 mentions) Ab3778, by Abcam (1 mentions) S 1000, by Vector Laboratories (1 mentions)

16 protocols using ab58655

Immunofluorescence Staining of Tendon and Cells

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Tendon sections were stained using a standardized procedure and incubated overnight at 4 °C with primary antibodies: anti-COL1A1 (Abcam, ab34710), anti-SCXA (Abcam, ab58655), and anti-TNMD (Abcam, ab203676). The sections were washed three times with PBS the next day and incubated with the secondary antibodies for 30 min at room temperature. 4′,6-Dimercapto-2-phenylindole (DAPI, Gibco, USA) was used for nuclear staining.
For cellular immunofluorescence (IF), cell slides were fixed with 4% paraformaldehyde (PFA) and incubated overnight at 4 °C with primary antibodies: anti-COL1A1 (Abcam, ab34710), anti-COL3A1 (Abcam, ab7778), anti-DCN (Affinity Biosciences, DF6543), and anti-TNMD (Abcam, ab203676). The slides were incubated with fluorescently conjugated secondary antibodies for 30 min at room temperature in the dark. DAPI was used for nuclear staining. The image was observed and captured with a fluorescence microscope (Olympus, Tokyo, Japan). ImageJ software was used for semi-quantitative analysis.

Yao Z., Li J., Xiong H., Cui H., Ning J., Wang S., Ouyang X., Qian Y, & Fan C. (2021). MicroRNA engineered umbilical cord stem cell-derived exosomes direct tendon regeneration by mTOR signaling. Journal of Nanobiotechnology, 19, 169.

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Tenogenic Differentiation of Stem Cells

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The TDSCs were plated on a density of 4×10⁵/well in a 60-mm dish and cultured in DMEM containing 10% FBS in the presence or absence IL-6 at the concentration of 10 ng/mL for 3 days and then lysed in SDS sample buffer. Scleraxis (Scx), tenomodulin (Tnmd), collagen 1 (Col1), collagen 3 (Col3) and GAPDH contents were examined by Immunoblot assays using the corresponding antibodies. The anti-scleraxis rabbit polyclonal antibody (Abcam, #ab58655) and anti-tenomodulin rabbit polyclonal antibody (Abcam, #ab203676) were purchased from Abcam (USA). The anti-collagen 1 rabbit polyclonal antibody (Proteintech, #14695-1-AP), anti-collagen 3 rabbit polyclonal antibody (Proteintech, #13548-1-AP), anti-Stat3 rabbit polyclonal antibody (Proteintech, #10253-2-AP) and anti-GAPDH rabbit polyclonal antibody (Proteintech, #10494-1-AP) were purchased from Proteintech (China). The anti-Phospho-Stat3(Tyr705) antibody (Affinity, #AF3295) were purchased from Affinity (China).

Chen S., Deng G., Li K., Zheng H., Wang G., Yu B, & Zhang K. (2018). Interleukin-6 Promotes Proliferation but Inhibits Tenogenic Differentiation via the Janus Kinase/Signal Transducers and Activators of Transcription 3 (JAK/STAT3) Pathway in Tendon-Derived Stem Cells. Medical Science Monitor : International Medical Journal of Experimental and Clinical Research, 24, 1567-1573.

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Immunostaining of Tenogenic Markers

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Before staining, the yarns with the hASCs were fixed and permeabilized with 10% formalin (Bio-Optica, Milan, Italy) and 0.1% X-100 Triton (ThermoFisher Scientific, Waltham, MA, USA), respectively. All the samples were blocked with a 2.5% normal horse serum (Vector Laboratories, Burlingame, CA, USA) for 1 h at room temperature (RT). The samples were then immunostained using primary antibodies against antiscleraxis (SCX, rabbit, ab58655, 1:200, Abcam, Cambridge, UK) and antitenomodulin (TNMD, rabbit polyclonal, 1:200) generated against the TNMD C-terminus (237-317 aa) provided by Prof. Denitsa Docheva (produced in cooperation with Metabion International, Planegg, Germany, PAB 201603-00002), and they were incubated overnight at 4 °C. The samples were then incubated with 0.3% hydrogen peroxide (H₂O₂, PanReac AppliChem, Castellar del Vallès, Spain) for 15 min at RT and then incubated with Alexa Fluor 488 fluorescent secondary antibody (anti-rabbit, A21206, 1:200, ThermoFisher Scientific, Waltham, MA, USA) for 1 h at RT in the dark. Finally, the cell nuclei and cytoskeleton were counterstained with DAPI (1:1000; Biotium, Fremont, CA, USA) and phalloidin (1:500; Sigma-Aldrich, St. Louis, MO, USA), respectively, at RT for 30 min. The immunolabeled samples (n = 4) were analyzed using confocal laser scanning microscopy (CLSM, TCS SP8, Leica, Wetzlar, Germany).

Graça A.L., Domingues R.M., Gomez-Florit M, & Gomes M.E. (2023). Platelet-Derived Extracellular Vesicles Promote Tenogenic Differentiation of Stem Cells on Bioengineered Living Fibers. International Journal of Molecular Sciences, 24(4), 3516.

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Histological and Immunofluorescence Analysis of Tendons

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Embedded tendons were cut into 5 μm sections and stained with hematoxylin-eosin (HE) and Masson’s trichrome reagent. For immunofluorescence experiments, sections were subjected to antigen retrieval and incubated overnight at 4 °C with anti-SCX (1:200, ab58655, Abcam, Cambridge, UK), anti-TNMD (1:200, ABIN872740, Sizhengbai Biotechnology, Suzhou, China), anti-COL1A1 (1:200, A1352, Abclonal), and anti-COL3A1 (1:200, A3795, Abclonal) antibodies, followed by the corresponding secondary antibodies conjugated with FITC or Cy3. Part of the tendon was freshly frozen in optimal cutting temperature compound for DHE staining. Visualization was performed using a fluorescent microscope and quantification using ImageJ software.

Ren X., Zhuang H., Zhang Y, & Zhou P. (2023). Cerium oxide nanoparticles-carrying human umbilical cord mesenchymal stem cells counteract oxidative damage and facilitate tendon regeneration. Journal of Nanobiotechnology, 21, 359.

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Histological Analysis of Murine Knee Joint

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Stifle joints were fixed in 4% paraformaldehyde for 24 h, decalcified with 14% ethylenediaminetetraacetic acid (EDTA) for 14 d, and embedded in OCT compound (Sakura, Torrance, California).Sagittal sections of the stifle joint were prepared at 6 µm thickness with a Cryofilm type 3c (SECTION-LAB, Hiroshima, Japan). Inguinal fat pads from donor Pdgfra orPdgfrβ‐CreER^T2‐mT/mG mice were also dissected, embedded in OCT, and cryosectioned at 30 μm thickness. Routine safranin O / fast greenstaining of stifle joints section was performed with methods adopted from past work^{25 (link)–27 (link)}.
For immunofluorescent immunohistochemistry, sections were washed in 1× PBS, blocked in 5% normal goat serum (S-1000, Vector Labs, CA, USA) for 30 min, and incubated with primary antibodies specific for ACAN (1:100, ab3778, Abcam, MA, USA), COL10(1:100, ab58632, Abcam), CD31 (1:100, ab 28364, Abcam)and scleraxis (1:100, ab58655, Abcam) at 4 °C overnight. Sections were then incubated with Alexa Fluor® 647-conjugated secondary antibodies (1:200, ab150083, Abcam) or DyLight® 594 (1:100, DI-1594, Vector Labs), and mounted with mounting medium containing DAPI (H-1500, Vector Labs, Burlingame, CA)(Supplemental Table 1). Immunofluorescence images were acquired on a Leica DM 6B or LSM 880 FCS confocal microscope (Carl Zeiss, Oberkochen, Germany).

Hsu G.C., Cherief M., Sono T., Wang Y., Negri S., Xu J., Peault B, & James A.W. (2021). Divergent effects of distinct perivascular cell subsets for intra-articular cell therapy in post-traumatic osteoarthritis. Journal of orthopaedic research : official publication of the Orthopaedic Research Society, 39(11), 2388-2397.

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Immunohistochemistry Analysis of Mouse Leg Tissue

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For IHC staining, the decalcified mouse leg tissue samples were prepared as above. Cryostat sectioning was performed on the frozen tissue samples to obtain about 5 µm-thick tissue sections, which were left at room temperature overnight. The tissue sections were fixed in 4% paraformaldehyde for 15 min and then the sections were incubated overnight at 4 °C with rabbit anti-α-SMA antibody (1:500; Cat. #ab124964, Abcam; Waltham, MA, USA), rabbit anti-TGF-β1 antibody (1:500; Cat. #ab215715, Abcam; Waltham, MA, USA), rabbit anti-Scx antibody (1:500; Cat. #ab58655, Abcam, Waltham, MA, USA), or rabbit anti-HMGB1 antibody (1:330; Cat. #ab18256, Abcam; Waltham, MA, USA). The positively stained results were tested using a rabbit-specific HRP/DAB IHC detection kit (Cat. #ab236466; Abcam, Waltham, MA, USA).
For AMPK activation testing, the fixed tissue sections were treated with 0.1% Triton X-100 at 37 °C for 30 min, then washed with PBS 3 times. The treated tissue sections were incubated with rabbit anti-AMPK antibody (1:500, Cat. #MA5-15815, ThermoFisher Scientific; Waltham, MA, USA) or with rabbit anti-phospho-AMPK antibody (1:500, Cat. #ab133448, Abcam; Waltham, MA, USA) at 4 °C overnight. The staining results were further tested using a rabbit-specific HRP/DAB IHC detection kit (Cat. #ab236466; Abcam, Waltham, MA, USA).

Zhang J., Brown R., Hogan M.V, & Wang J.H. (2023). Mitigating Scar Tissue Formation in Tendon Injuries: Targeting HMGB1, AMPK Activation, and Myofibroblast Migration All at Once. Pharmaceuticals, 16(12), 1739.

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Immunohistochemical Analysis of Tenascin C and Scleraxis

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Immunohistochemistry was performed using rabbit anti-human tenascin C monoclonal (ab108930; Abcam, Cambridge, UK) or rabbit anti-human scleraxis polyclonal (ab58655; Abcam) antibodies. Tissue sections on slides were deparaffinized, incubated with methanol and 0.3% hydrogen peroxide (H₂O₂) for 30 min, and washed. The slides were then incubated with the respective primary antibodies (1:200) at 4 °C overnight. After additional washing steps, the slides were further incubated with horseradish-conjugated secondary polyclonal rabbit immunoglobulin G antibodies (ImmPRESS reagent anti-rabbit Ig kit; Vector Laboratories Burlingame, CA, USA). After additional washing steps, immunoreactivity was visualized using a diaminobenzidine peroxidase substrate kit (Vector Laboratories), followed by counterstaining with hematoxylin. Negative controls were samples immunostained without the primary antibody.

Nakanishi Y., Okada T., Takeuchi N., Kozono N., Senju T., Nakayama K, & Nakashima Y. (2019). Histological evaluation of tendon formation using a scaffold-free three-dimensional-bioprinted construct of human dermal fibroblasts under in vitro static tensile culture. Regenerative Therapy, 11, 47-55.

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Western Blot Analysis of Extracellular Matrix Proteins

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Western blot assay was conducted for determination of Col I, Col III, α-SMA, Scx, TnC and TGF-β1. Similar to the procedure of determine of exosome proteins, samples were separated with 10% SDS-PAGE, transferred onto a PVDF membrane, blocked by non-fat milk for 1 h at room temperature and incubated with the following primary antibodies at 4 °C overnight: anti-β-actin (ab8227, Abcam) anti-Col I (ab34710, Abcam), anti-Col III (ab7778, Abcam), anti-α-SMA (ab32575, Abcam), anti-Scx (ab58655, Abcam) and anti-TnC (ab108930, Abcam), TGF-β1 (ab92486, Abcam). The samples were then incubated with corresponding secondary antibody (Goat Anti-Rabbit IgG H&L, ab205718, Abcam) at 37 °C for 45 min. β-actin was used as the internal control. Protein brands were visualized by Super Signal West Pico Chemiluminescent Substrate kit (Pierce; Thermo Fisher Scientific, Inc., Waltham, MA, USA), and the ImageJ software (Rasband; NIH, USA) was used for quantification of the bands to assess relative proteins expression.

Li J., Liu Z.P., Xu C, & Guo A. (2020). TGF-β1-containing exosomes derived from bone marrow mesenchymal stem cells promote proliferation, migration and fibrotic activity in rotator cuff tenocytes. Regenerative Therapy, 15, 70-76.

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Protein Expression Analysis of Tendon Cells

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Total proteins were extracted using pre-cooled RIPA lysis buffer (Beyotime, Beijing, China), then separated by 10% SDS-PAGE gels, followed by shifting onto PVDF membranes. Then, the membranes were probed with primary antibodies at 4 °C for 12 h and then HRP-conjugated antibodies for 2 h at 37 °C. The primary antibodies included Scleraxis (SCX) (1:2000, ab58655), mohawk homeobox (MKX) (1:2000, ab236400), Collagen1A1 (COL1A1) (1:1000, ab34710), Fibromodulin (FMOD) (1:1000, ab267465) and GAPDH (1:100, ab181602) and were obtained from Abcam (Cambridge, MA, USA). The ECL procedure (Merck Millipore) was adopted for proteins observation, and the gray value was evaluated using ImageJ soft (National Institutes of Health, Bethesda, MD, USA).

Yang G., Chen F., Zhang C, & Gu C. (2023). Circ_0005736 promotes tenogenic differentiation of tendon-derived stem cells through the miR-636/MAPK1 axis. Journal of Orthopaedic Surgery and Research, 18, 660.

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Characterization of Isolated Tenocytes

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For characterization of isolated tenocytes, immunocytochemistery was perfrormed. The tenocytes were rinsed twice with PBS followed by fixation using paraformaldehyde 4% in PBS (pH=7.2) for 20 minutes at 25°C. After removing the fixative solution, the cells were washed with PBS for 3 A c c e p t e d M a n u s c r i p t times and permeabilized with 0.2% (v/v) Triton X-100 in PBS for 40 minutes. Following that, cells were washed using PBS (3 times) and incubated with BSA 1% in PBS at 37°C for 1 h to block non-specific antigen-antibody reactions. Next, they were incubated with primary antibodies against scleraxis (1:250, ab58655, Abcam, UK) and Tenomodulin (1:200, orb101154, Biorbyt, UK) at 4°C overnight. Finally, anti SCX treated samples were incubated with Alexa Fluor® 647conjugated secondary antibody (1:300, ab150079, Abcam, UK) and Anti tenomodulin treated samples were incubated with Alexa Fluor® 488-conjugated secondary antibody (1:300, ab150077, Abcam, UK) for 1 h in the darkness at 25°C. DAPI staining (1µg/ML, Sigma) was used for nuclei detection. After immunocytochemistry, the samples were imaged using fluorescent microscopy (Olympus, Japan). Then by dividing the cells expressing TNMD (as a specific marker of adult tenocytes) by the total number of cells, it was shown that in different samples, on average, 93 % of cells expressed TNMD.

Haramshahi S.M.A., Bonakdar S., Moghtadaei M., Kamguyan K., Thormann E., Tanbakooei S., Simorgh S., Brouki-Milan P., Amini N., Latifi N., Joghataei M.T., Samadikuchaksaraei A., Katebi M, & Soleimani M. (2020). Tenocyte-imprinted substrate: a topography-based inducer for tenogenic differentiation in adipose tissue-derived mesenchymal stem cells. Biomedical materials (Bristol, England), 15(3).

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