Intestinal alkaline phosphatase, LPS (E coli serotype 055:B5), and Ripa buffer were purchased from Sigma-Aldrich, and TRIzol was purchased from Invitrogen. The iScriptReverse Transcription Supermix for RT-qPCR and iQ SYBR Green Supermix Kit were obtained from BIO-RAD, and the Coomassie (Bradford) Blue Protein Assay Kit was from Fisher Scientific. Goat anti-human ZO-1 and rabbit anti-human Occludin were purchased from Santa Cruz Biotechnology, and Alexa Flour secondary antibodies were obtained from Lifetechnology.
Alexa flour secondary antibody
Manufactured by Thermo Fisher Scientific
Sourced in United States
Alexa Fluor secondary antibodies are a range of fluorescently-labeled secondary antibodies manufactured by Thermo Fisher Scientific. These antibodies are designed to detect and visualize primary antibodies in various immunodetection techniques, such as immunofluorescence microscopy and flow cytometry. The Alexa Fluor dyes provide bright, photostable fluorescence signals to enable sensitive and reliable detection of target proteins.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 510, by Zeiss (4 mentions)
Dapi containing mounting medium, by Vector Laboratories (2 mentions)
Lps e coli serotype 055 b5, by Merck Group (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
Iq sybr green supermix kit, by Bio-Rad (1 mentions)
Iscript reverse transcription supermix for rt qpcr, by Bio-Rad (1 mentions)
Anti phospho erk1 2, by Cell Signaling Technology (1 mentions)
Hoechst, by Merck Group (1 mentions)
Mca4095, by Bio-Rad (1 mentions)
Ab9610, by Merck Group (1 mentions)
Normal donkey serum (nds), by Merck Group (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Fluoromount g, by Southern Biotech (1 mentions)
Gtpγs, by Merck Group (1 mentions)
Rab10, by Cell Signaling Technology (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Optiprep gradient solution, by Merck Group (1 mentions)
Histone h3, by Cell Signaling Technology (1 mentions)
Insulin, by Merck Group (1 mentions)
15 protocols using alexa flour secondary antibody
1
Intestinal Barrier Integrity Assessment
2
Immunostaining of Formalin-Fixed Samples
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Immunostaining was performed on formalin-fixed paraffin embedded sections as previously described [6 (link)]. IF utilized Alexa-Flour secondary antibodies (Life Technologies) with DAPI (100 ng/ml) for DNA counterstaining. IHC was completed with EnVision HRP antibody system (Dako, Carpinteria, CA). Antibodies: anti-FLAG pAb, 1:1000 (Sigma); anti-loricrin mAb, 1:1000 (Covance); anti-β-gal pAb 1:5000 (MP Biomedicals, Santa Ana, CA); 5E1 anti-Shh mAb 1:50 (DSHB, Iowa City, IA); anti-Phospho-Erk1/2 1:400 (Cell Signaling, Danvers, MA).
3
Immunocytochemistry of Oligodendrocyte Markers
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After siRNA treatment, cells were blocked with 5% normal donkey serum (NDS) (Sigma‐Aldrich) supplemented with 0.1% Triton X‐100 (Sigma‐Aldrich) in PBS for 1 hr at RT. Then cells were incubated for 1 hr at RT with primary antibodies (Mouse anti‐CNPase antibody, 1:500 (C5922, Sigma‐Aldrich); Rabbit anti‐Olig2 antibody, 1:500 (AB9610, Millipore); or rat anti‐MBP antibody, 1:500 (MCA4095, Serotec)) in 5% NDS with 0.1% Triton X‐100. After three washes, the cells were incubated with the appropriate Alexa Flour secondary antibodies (1:500, Life‐technologies) diluted in 5% NDS with 0.1% Triton X‐100 for 1 hr at RT. Nuclei were stained with Hoechst (2μg/ml, Sigma‐Aldrich) for 10 min at RT, before the coverslips were mounted using Fluoromount G (Southern Biotech).
4
Immunofluorescent Staining of Tissues and Cells
5
Immunoblotting Analysis of Insulin Signaling Proteins
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Samples were run on 4–20% precast SDS–PAGE gels (Novex, Life Technologies) and transferred to nitrocellulose membrane. Antibodies to HA and Myc (monoclonal mouse and polyclonal rabbit) and human Rlf were purchased from Santa Cruz Biotechnology. Mouse-FLAG antibodies were from Agilent Technologies. Anti-RFP antibodies were from Abcam (Cambridge, MA). AS160 and pAS160 (T642) antibodies were from EMD Millipore. Rabbit anti-Rlf serum described previously (Wolthuis et al., 1997 (link)) was affinity purified (Pacific Immunologies, Ramona, CA) and used for immunoblotting. RalA and caveolin3 antibodies were from BD Biosciences (San Jose, CA). Transferrin receptor antibody was from Zymed Labs. Glut4 and Sec8 antibodies were purchased from Alpha Diagnostics (San Antonio, TX) and Enzo LifeSciences (Farmingdale, NY). Rab10, Akt, pAkt (S473), pAkt-substrate, histone H3, and IRAP antibodies were from Cell Signaling (Danvers, MA). Horseradish peroxidase–conjugated secondary antibodies were obtained from Thermo Scientific (Waltham, MA), and Alexa Flour secondary antibodies were from Molecular Probes, Life Technologies. Insulin, GDP, GTPγS, and Optiprep gradient solution were purchased from Sigma-Aldrich (St. Louis, MO).
6
Multicolor Immunofluorescence Labeling
7
Immunofluorescence Staining of Brain Tissue
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Immunofluorescence staining was performed according to described previously (Li et al., 2022 (link)). Briefly, mice were perfusion-fixed with 4% paraformaldehyde and soaked in PFA for 48 h, then brian was replaced with sucrose, and 30um brain tissue sections were prepared. Sections were washed with PBS for 15 min (5 min × 3). After washing, the sections were blocked with blocking buffer (10% normal goat serum in 0.3% Triton X-100 in PBS) for 1 h at room temperature and then incubated with primary antibodies overnight at 4°C, Rabbit anti-Iba1 (1: 1000, Wako,#019–19,741); Mouse anti-GFAP (1,1,000, Sigma-Aldrich, #MAB360). The next day, sections were incubated with secondary antibodies (Alexa Flour secondary antibodies, Thermo Fisher) at room temperature for 1 h; the hoechst was applied in the last 10 min. The sections were washed with PBS for 15 min (5 min × 3). After washing, sections were transferred onto glass slides, and glass coverslips were mounted using a mounting medium. Images were captured using a confocal microscope and were analyzed by ImageJ software.
8
Brain Tissue Immunofluorescence Staining
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Immunofluorescence staining was performed according to previously reported protocols [56 (link)]. Briefly, brain tissue sections (20-μm thick) were washed with PBS for 15 min (5 min × 3). After washing, the sections were treated with blocking buffer (10% goat serum in 0.3% Triton X-100 in PBS) for 1 h at room temperature. After blocking, the tissue was treated with primary antibodies (Iba1, GFAP) for overnight at 4̊ °C. The next day, secondary antibodies (Alexa Flour secondary antibodies, ThermoFisher) were applied at room temperature for 1 h. The sections were washed with PBS for 5 min three times. After washing, the sections were transferred to slides, and glass coverslips were mounted using the mounting medium. The images were taken under inverted fluorescence microscope I X73 Olympus.
9
Immunofluorescence Staining of Cell Lines
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Cell lines were grown at 1 × 104 cells/cm2 on PDL‐coated glass coverslips for 48 h, fixed with 4% paraformaldehyde, and exposed to primary antibodies O/N at 4°C and to secondary antibodies for 2 h at RT. The following antibodies were used: PECAM (polyclonal rabbit, 1:20, Abcam), PAI‐1 (polyclonal rabbit, 1:50, Sigma Prestige), SNAI1/2 (polyclonal rabbit, 1:500, Abcam), S100A4 (monoclonal mouse, 1:80, Sigma), αSMA (monoclonal rabbit, 1:500, Abcam; monoclonal mouse, 1:100, Sigma), and AlexaFlour secondary antibodies (1:1500, ThermoFisher Scientific). Hoechst 33342 was used to label cell nuclei. Slides were imaged using a Zeiss Axio Imager M2 microscope.
10
Immunofluorescence Staining of Brain Tissue
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Immunofluorescence staining was performed according to previously reported protocols (Shah et al., 2017 (link)). Briefly, brain tissue sections (20 μm thick) were washed with PBS for 15 min (5 min × 3). After washing, the sections were treated with blocking buffer (10% Goat serum in 0.3% Triton X–100 in PBS) for 1 h at room temperature. After blocking the tissue was treated with primary antibodies with a dilution ratio of 1:500 μL (Iba1, GFAP) for overnight at 4°C. Next day secondary antibodies 1:400 μL (Alexa Flour secondary antibodies, ThermoFisher, Waltham, MA, United States) were applied at room temperature for 1 h. The sections were washed with PBS for 5 min three times. After washing, sections were transferred to slides, and glass coverslips were mounted using the mounting medium. The images were taken under inverted fluorescence microscope IX73 Olympus. ImageJ software was used to quantify the relatively integrated density of GFAP and Iba-1.
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