Alexa fluor 546 conjugated anti rabbit igg

Manufactured by Thermo Fisher Scientific

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Alexa Fluor 546-conjugated anti-rabbit IgG is a secondary antibody that binds to rabbit primary antibodies. The Alexa Fluor 546 dye provides a fluorescent label for detection and visualization purposes.

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11 protocols using alexa fluor 546 conjugated anti rabbit igg

Immunofluorescent Staining of TNIP1 in HPV-KER and Skin Cells

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HPV-KER cells were grown on glass sides, fixed with 2% paraformaldehyde (PFA) for 5 min, permeabilized with 0.1% Triton X, 2% PFA-containing phosphate-buffered saline (PBS), and blocked for 2 h at room temperature with PBS containing 1% bovine serum albumin (BSA), 0.05% Triton X 100, and 10% goat serum. Cells were stained overnight at 4°C with anti-human TNIP1 antibody (Sigma Aldrich, St. Louis, MO, United States) or rabbit IgG for isotype control. As a secondary antibody, Alexa Fluor 488 conjugated anti-rabbit IgG, was used for 2 h. Filamentous actin was stained by Alexa Fluor 546® phalloidin (Life Technologies, Carlsbad, United States) diluted 1:100 in PBS containing 1% BSA for 20 min. Nuclei were stained for 10 min with 4′,6-diamidino-2-phenylindole (DAPI) diluted 1:500.
Frozen sections of ex vivo skin models were pre-incubated with PBS for 5 min and fixed and permeabilized with Foxp3 staining buffer set (Thermo Scientific, Rockford, United States) and blocked for 1 h at room temperature with TBS containing 1% BSA and 1% normal goat serum. Cells were stained for 1 h with anti-human TNIP1 antibody or rabbit IgG for isotype control. As a secondary antibody, Alexa Fluor 546 conjugated anti-rabbit IgG (Thermo Scientific, Rockford, United States), was used for 1 h at room temperature. Nuclei were stained for 6 min with DAPI diluted 1:100.

Erdei L., Bolla B.S., Bozó R., Tax G., Urbán E., Kemény L, & Szabó K. (2018). TNIP1 Regulates Cutibacterium acnes-Induced Innate Immune Functions in Epidermal Keratinocytes. Frontiers in Immunology, 9, 2155.

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Immunofluorescence Staining and Flow Cytometry

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Cells were fixed with 4% paraformaldehyde
and blocked in PBS with 5% skim milk and 0.2% Triton X-100 at 4 °C
for 30 min, respectively. Then, the cells were reacted at room temperature
for 2 h with the antibodies of anti-GFAP mouse monoclonal antibody
(1:1000, Merck Millipore) or anti-pSTAT3 rabbit monoclonal antibody
(1:500, Cell Signaling Technology, Danvers, MA), followed by reaction
at 4 °C for 30 min with Alexa Fluor 647-conjugated anti-mouse
IgG (1:1000; Thermo Fisher Scientific) or Alexa Fluor 546-conjugated
anti-rabbit IgG (1:1000; Thermo Fisher Scientific), respectively.
The cells were then subjected to flow cytometry analysis with CytoFLEX
S (Beckman Coulter, Brea, CA, Japan).

Otsuka S., Kawamura M., Fujino S., Nakamura F., Arai D., Fusetani N, & Nakao Y. (2022). Coronarin D, a Metabolite from the Wild Turmeric, Curcuma aromatica, Promotes the Differentiation of Neural Stem Cells into Astrocytes. Journal of Agricultural and Food Chemistry, 70(10), 3300-3309.

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Immunofluorescence Staining of TNFAIP3 in Ex Vivo Skin

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Frozen sections of ex vivo skin models were pre-incubated with PBS for 5 min, fixed with 4% paraformaldehyde for 5 min and permeabilized with 0.25% TritonX-100 (Thermo Scientific, Rockford, USA) containing Tris-buffered saline (TBS) for 10 min. For blocking, TBS containing 5% foetal bovine serum (EuroClone, Milan, Italy) and 5% normal goat serum (Sigma Aldrich, St Louis, MO, USA) was used for 1 h at room temperature. Cells were stained for 1 h with anti-human TNFAIP3 antibody diluted 1:300 or rabbit IgG (Santa Cruz Biotechnology) for isotype control. As a secondary antibody, Alexa Fluor 546 conjugated anti-rabbit IgG (Thermo Scientific) was used for 1 h at room temperature. Nuclei were stained for 5 min with 4′,6-diamidino-2-phenylindole (DAPI) diluted 1:100. TNFAIP3 was visualized using a ZEISS LSM 880 Confocal Laser Scanning Microscope (Zeiss, Oberkochen, Germany), magnification × 63.

ERDEI L., BOLLA B.S., BOZÓ R., TAX G., URBÁN E., BURIÁN K., KEMÉNY L, & SZABÓ K. (2021). Tumour Necrosis Factor Alpha-induced Protein 3 Negatively Regulates Cutibacterium acnes-induced Innate Immune Events in Epidermal Keratinocytes. Acta Dermato-Venereologica, 101(1), 1470.

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Immunohistochemistry for MCP-1 Expression

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Formalin‐fixed paraffin‐embedded tissue specimens were processed according to standard protocols. For the detection of MCP‐1‐producing cells, sections were incubated with primary antibodies of MCP‐1(73680; Abcam, Cambridge, MA)at 1:100 dilution. Immunohistochemical staining was performed by an autoimmunostainer with biotin blocking to remove endogenous hepatic biotin. The fuchsin substrate‐chromogen system (K0624; DAKO, Denmark) was used for development. LX‐2 cells in culture were fixed with 4% paraformaldehyde for 15 minutes followed by briefly permeabilizing with 0.2 % Triton X‐100. Dishes were incubated with a primary antibody for 1 hour followed by incubation with an appropriate secondary antibody for an additional hour. The primary antibodies were as follows MCP‐1(73680; Abcam), 1:200 dilution; MCP‐1 (505905; BioLegend, San Diego, CA), 1:500 dilution; C‐C chemokine receptor type 2(21667; Abcam), 1:1,000 dilution; and type IV collagen (c1926; Sigma), 1:200 dilution. The secondary antibodies were as follows Alexa Fluor 488‐conjugated anti‐mouse IgG (H+L) (A‐11001; Molecular probes, Carlsbad, CA), Alexa Fluor 546‐conjugated anti‐rabbit IgG (A‐11035; Molecular Probes), and Alexa Fluor 546‐conjugated anti‐hamster IgG (H+L) (A‐21111; Molecular Probes). The nucleus was stained with 4′,6‐diamidino‐2‐phenylindole.

Kobayashi K., Yoshioka T., Miyauchi J., Nakazawa A., Kiyokawa N., Maihara T, & Usami I. (2018). Role of monocyte chemoattractant protein‐1 in liver fibrosis with transient myeloproliferative disorder in down syndrome. Hepatology Communications, 2(3), 230-236.

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Immunohistochemical Analysis of Bone Markers

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The cultured femurs were fixed in neutral buffered formaldehyde for 24 h, followed by decalcification with a 0.5 M EDTA-2Na solution (pH 7.4) for 7 days. Specimens were dehydrated through a graded series of ethanol, embedded into paraffin, and then transversely cut into 5-µm-thick sections. Deparaffinized sections were blocked with normal goat serum (ThermoFisher Scientific, Waltham, MA, USA) to block the non-specific antibody binding sites. The specimens were then incubated with mouse anti cathepsin D (Abcam, Cambridge, UK), rabbit anti MMP10 (Abcam, Cambridge, UK), mouse anti ALP (Novus Biologicals, Littleton, CO, USA), and rabbit anti osterix (Abcam, Cambridge, UK) antibodies. The secondary antibodies were as follows: Alexa Fluor 546-conjugated anti rabbit IgG (Molecular Probes, ThermoFisher Scientific, Waltham, MA, USA), Alexa Fluor 488-conjugated anti mouse IgG (Molecular Probes, ThermoFisher Scientific, Waltham, MA, USA). Nuclei were stained with DAPI (ThermoFisher Scientific, Waltham, MA, USA).

Matsugaki A., Harada T., Kimura Y., Sekita A, & Nakano T. (2018). Dynamic Collision Behavior Between Osteoblasts and Tumor Cells Regulates the Disordered Arrangement of Collagen Fiber/Apatite Crystals in Metastasized Bone. International Journal of Molecular Sciences, 19(11), 3474.

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Histological Analysis of Femur Tissue

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The cultured femurs were fixed in neutral buffered formaldehyde for 24 h, followed by decalcification using 0.5 M EDTA-2Na solution (pH 7.4) for 7 d. Specimens were dehydrated through a graded series of ethanol, embedded into paraffin, and transversely cut into 5 μm-thick sections. Deparaffinized sections were incubated with normal goat serum (Thermo Fisher Scientific, Waltham, MA, USA) to block non-specific antibody binding sites. The specimens were then incubated with rabbit anti MMP1 (Abcam, Cambridge, UK), mouse anti melanA (Santa Cruz, Dallas, TX, USA), mouse anti ALP (Novus Biologicals, USA, Centennial, CO, USA), and rabbit anti Cx43 (Cell Signaling, Danvers, MA, USA) antibodies. The secondary antibodies used were as follows: Alexa Fluor 546-conjugated anti-rabbit IgG (Molecular Probes, Thermo Fisher Scientific, Waltham, MA, USA), and Alexa Fluor 488-conjugated anti-mouse IgG (Molecular Probes, Thermo Fisher Scientific, Waltham, MA, USA). Nuclei were stained with DAPI (Thermo Fisher Scientific, Waltham, MA, USA).

Matsugaki A., Kimura Y., Watanabe R., Nakamura F., Takehana R, & Nakano T. (2021). Impaired Alignment of Bone Matrix Microstructure Associated with Disorganized Osteoblast Arrangement in Malignant Melanoma Metastasis. Biomolecules, 11(2), 131.

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Bortezomib Induces Autophagy in Cancer Cells

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After transient transfection with GFP-LC3 or GFP-RFP-LC3, cancer cells were cultured on chamber slide at the concentration of 10³ cells per well overnight. After treatment with bortezomib overnight, cells were fixed with acetone for 5 min and incubated in blocking buffer (5% normal goat serum in PBS) for 1 h at RT to reduce nonspecific binding. For LAMP-2 (Sigma, PRS3627) or cathepsin B (Biovision, 3190-100) staining, cells were incubated with a rabbit polyclonal antibody (1 : 100; Invitrogen, Grand Island, NY, USA; A22283-300L) overnight. After being incubated with Alexa Fluor 546 conjugated anti-rabbit IgG (1 : 100; Invitrogen, A22283-300L), the slides were mounted with mounting medium (SouthernBiotech, Birmingham, AL, USA; 0100-20) and cover glass, and analyzed with the Leica TCS SP2 laser-scanning confocal system (Leica, Wetzlar, Germany) as described previously.^{61 (link)}

Kao C., Chao A., Tsai C.L., Chuang W.C., Huang W.P., Chen G.C., Lin C.Y., Wang T.H., Wang H.S, & Lai C.H. (2014). Bortezomib enhances cancer cell death by blocking the autophagic flux through stimulating ERK phosphorylation. Cell Death & Disease, 5(11), e1510-.

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Immunolocalization of TcCP30 in Tribolium Pupae

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To analyze protein localization of TcCP30, immunostaining was performed as described previously37 (link). Cryosections (~12 μm) of day 5 pupae that had been injected with dsTcCP30 or dsTcVer (200 ng per insect) at the late larval stage were prepared. The tissues were rinsed with PBST (0.01 M PBS, pH 7.4 containing 0.2% TWEEN 20), and then blocked with blocking buffer (PBS containing 2% bovine serum albumin) for 1 h at room temperature. Tissues were incubated with the anti-TcCP30 antibody (1:300 in blocking buffer) for 3 h at room temperature followed by washing with PBST three times for 5 min each. Then the tissues were incubated with Alexa Fluor 546-conjugated anti-rabbit IgG (Invitrogen) (1:300 in blocking buffer) as secondary antibody for 1 h at room temperature. After washing the tissues three times with PBST, the FITC-conjugated chitin binding domain probe (FITC-CBD, 1:300 in PBST)36 (link) was applied and incubated at 4 °C overnight. The sections were washed with PBST three times for 5 min each at room temperature and then nuclei were stained with TO-PRO3 (1:1000 in PBST) (Invitrogen) for 1 h at room temperature. Tissues were observed using a confocal laser-scanning microscope (Olympus FV500) equipped with appropriate filters.

Mun S., Young Noh M., Dittmer N.T., Muthukrishnan S., Kramer K.J., Kanost M.R, & Arakane Y. (2015). Cuticular protein with a low complexity sequence becomes cross-linked during insect cuticle sclerotization and is required for the adult molt. Scientific Reports, 5, 10484.

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Immunohistochemical Analysis of Microglia

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Microglia were immunostained for Iba-1 detection as previously described (40 (link)). Briefly, animals were deeply anesthetized and transcardially perfused with sterile PBS (pH 7.4) and 10% formalin. Brains were removed, post-fixed in formalin for 2 days and incubated in 30% sucrose for cryoprotection. Brains were sectioned (25μm) and brain regions were located anatomically in accordance with the stereotaxic rat brain atlas (Paxinos and Watson, 2004). Hippocampal brain sections were incubated overnight at 4°C with IBA1 antibody (1:1000, Wako), washed several times and incubated overnight at 4°C in secondary antibody Alexa Fluor 546-conjugated anti-rabbit IgG (1:10000; Invitrogen). Sections were then mounted and kept at 4°C until processed.

Franklin T.C., Wohleb E.S., Zhang Y., Fogaça M., Hare B, & Duman R.S. (2017). Persistent increase in microglial RAGE contributes to chronic stress Induced priming of depressive-like behavior. Biological psychiatry, 83(1), 50-60.

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Quantitative Analysis of Bone Repair

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Mice were euthanized with a lethal dose of isoflurane. Thighs were dissected, fixed with 10% neutral-buffered formalin, and subjected to μCT imaging (Scan Xmate-L090; Com Scan Techno). For volumetric quantification, longitudinal sections through the segmental defect were analyzed using ImageJ software to calculate percentage of callus formation. After radiologic assessment, explants were embedded in SCEM compound (Leica Microsystems) and quick-frozen. Frozen specimens were sectioned into 6-μm slices and subjected to H&E and Alizarin Red S staining, then fixed further with 4% paraformaldehyde and blocked with Blocking One Histo (Nacalai Tesque) and goat anti-mouse IgG (Abcom) before immunostaining with mouse anti-human OC (Bio-Rad) and rabbit anti-human vimentin (Abcam) antibodies and DAPI. For visualization of osteocalcin and vimentin, cryosections were incubated with Alexa Fluor 488-conjugated anti-mouse IgG (Invitrogen) and Alexa Fluor 546-conjugated anti-rabbit IgG (Invitrogen) antibodies, respectively. Fluorescence and optical light microscopic images were obtained and analyzed with BZ-X710 and BZ-II Analyzer software (Keyence).

Yamamoto K., Kishida T., Nakai K., Sato Y., Kotani S.I., Nishizawa Y., Yamamoto T., Kanamura N, & Mazda O. (2018). Direct phenotypic conversion of human fibroblasts into functional osteoblasts triggered by a blockade of the transforming growth factor-β signal. Scientific Reports, 8, 8463.

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