For western blot analysis, cells were harvested at 60–70% confluency and lysed with Laemmli buffer (2× concentrated) with 2 mM PMSF, 10 μg/ml aprotinin, 10 μg/ml leupeptin, 5 mM EGTA, 1 mM EDTA, 2 mM Na4P2O7, 5 mM NaF, and 5 mM Na3VO4. Samples with equal amounts of protein were loaded per well, resolved in SDS–PAGE and then transferred onto nitrocellulose membrane. The membranes were blocked for 1 h in 5% skimmed milk and probed with specific primary antibodies at 4°C. Appropriate secondary Alexa Fluor®-conjugated antibodies (680 or 790 nm) (Jackson ImmunoResearch, #111-625-144, #715-655-150) and Odyssey® CLx imaging system (LI-COR® Biosciences) were used to detect protein bands.
Alexa fluor conjugated antibody
Manufactured by Jackson ImmunoResearch
Sourced in United States
Alexa Fluor-conjugated antibodies are fluorescent dye-labeled antibodies. They are designed for use in various immunodetection techniques, such as flow cytometry, immunofluorescence, and Western blotting. The Alexa Fluor dyes provide bright, photostable fluorescence that can be detected using common fluorescence detection systems.
Automatically generated - may contain errors
Lab products found in correlation
Eclipse e800 microscope, by Nikon (4 mentions)
Las af, by Leica camera (3 mentions)
Wide field fluorescence microscope, by Leica camera (3 mentions)
Ab11822, by Abcam (2 mentions)
Custom stellaris fish probes smf 1000 series quasar 570, by Biosearch Technologies (2 mentions)
Smooth muscle actin, by Merck Group (2 mentions)
Vectashield with 4 6 diamidino 2 phenylindole, by Vector Laboratories (2 mentions)
Odyssey clx imaging system, by LI COR (1 mentions)
Ab5392, by Abcam (1 mentions)
Ab133350, by Abcam (1 mentions)
Donkey serum, by Jackson ImmunoResearch (1 mentions)
Mouse anti smi32, by BioLegend (1 mentions)
Rat anti mbp, by Abcam (1 mentions)
Goat anti cd206, by Abcam (1 mentions)
Fluoroshield with dapi, by Merck Group (1 mentions)
Rabbit anti iba1, by Fujifilm (1 mentions)
Oct compound, by Sakura Finetek (1 mentions)
Chamber slide, by Thermo Fisher Scientific (1 mentions)
P36931, by Thermo Fisher Scientific (1 mentions)
11 protocols using alexa fluor conjugated antibody
1
Western Blot Protein Detection Protocol
2
Immunofluorescence Staining of Neuronal Markers
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Cells were fixed in 4% paraformaldehyde for 1 h, then permeabilized and blocked with 0.1% of Triton X-100 and 5 % donkey serum (Jackson ImmunoResearch Laboratories, West Grove, PA) to reduce nonspecific background. After an overnight incubation at 4°C with the primary antibodies [anti-β-tubulin III (StemCell Technology, 60100AD), anti-Map2a (Abcam, ab5392), and anti-Thy1 (Abcam, ab133350)], cells were washed with PBS and incubated with secondary AlexaFluor-conjugated antibodies appropriate for the species (Jackson ImmunoResearch Laboratories, 1:500).
3
Immunofluorescence and RNA FISH Analysis of Nuclear Bodies
4
Immunohistochemical Analysis of ICH in Mice
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Mice were euthanized with sodium pentobarbital and perfused with 4% paraformaldehyde in 0.1 mM PBS (pH 7.4). Brains were harvested and cryoprotected in 30% (wt/vol) sucrose solution for 24 h. The brain tissues were then frozen with OCT compound (Sakura Finetek, Inc., Torrance, CA, USA). The sections were blocked with 10% (vol/vol) normal donkey serum for 1 h, followed by overnight incubation (4 °C) with the following primary antibodies: rat anti-MBP (Abcam, Cambridge, UK), mouse anti-SMI32 (BioLegend, San Diego, CA, USA), rabbit anti-Iba1 (Wako, Tokyo, Japan), goat anti-CD206 (Abcam), and rat anti-CD16/32 (Abcam). All the primary antibodies used are listed in Table S2 . The appropriate Alexa Fluor-conjugated antibodies (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) were used as secondary antibodies. Finally, the fluoroshield with DAPI (Sigma-Aldrich, St. Louis, MO, USA) was used to mount the cover slides. Cell numbers were calculated per square millimeter from 2 random microscopic fields on 4 sections (a total of eight images) cut through the STR. All procedures were performed in a blinded fashion. The fluorescence intensity of MBP and SMI32 was measured to demonstrate axonal damage after ICH.
5
Immunofluorescence Localization of SMN, Coilin, and TCAB1
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Immunofluorescence experiments were carried out on cells grown on coverslips. Cells were fixed with 4% PFA and permeabilized with 0.5% Triton X-100 in PBS. Coverslips were incubated with primary antibodies in 3% BSA for 1 h at room temperature: monoclonal anti-SMN (05-1532 Sigma-Aldrich clone 2B1, 1:1000); rabbit anti-Coilin (H300 Santa Cruz SC-32860, 200 μg/ml); rabbit anti-TCAB1 ((49 (link)), 25 ng/ml). Coverslips were washed three times with PBS and incubated with secondary Alexa Fluor-conjugated antibodies (Jackson Immunoresearch). Coverslips were washed three times in PBS, counterstained in a 300 nM 4′,6-diamidino-2-phenylindole (DAPI) solution and mounted in ProLong Gold Anti-fade Mountant. Images were captured on a Leica wide-field fluorescence microscope and processed using Leica LAS AF and Photoshop.
6
Immunofluorescence Staining of FLAG-tagged Cells
7
Immunofluorescence and RNA FISH Analysis of Nuclear Bodies
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IF experiments were carried out on cells grown on coverslips. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.5% Triton X-100 in PBS. Coverslips were incubated with primary antibodies in 3%BSA for 1 hr at room temperature: anti-Coilin (Abcam, AB11822, 25ng/mL); anti-TCAB1 (Venteicher et al., 2009 (link), 25 ng/mL). Coverslips were washed 3x with PBS and incubated with secondary Alexa Fluor conjugated antibodies (Jackson Immunoresearch). Coverslips were washed 3x in PBS, counterstained in a 300 nM DAPI solution and mounted in ProLong Gold Anti-fade Mountant.
RNA FISH experiments to detect hTR, U93scaRNA, U2 snRNA or U13 snoRNA were performed with Custom Stellarisâ FISH Probes (SMF-1000 Series; Quasar 570 conjugated, 75 nM working concentration) from Biosearch Technologies, following the manufacturer’s protocol. Probe sequences are listed in theKey Resources Table . Coverslips were mounted in 2x SSC for analysis. Images were captured on a Leica wide-field fluorescence microscope and processed using Leica LAS AF and Photoshop. Error bars, S.E.M, and significance were calculated by one-way ANOVA with GraphPad Prism.
RNA FISH experiments to detect hTR, U93scaRNA, U2 snRNA or U13 snoRNA were performed with Custom Stellarisâ FISH Probes (SMF-1000 Series; Quasar 570 conjugated, 75 nM working concentration) from Biosearch Technologies, following the manufacturer’s protocol. Probe sequences are listed in the
8
Quantifying Myocardial Microvasculature
9
Myocardial Notch Signaling Pathway
10
Quantifying Myocardial Vascular Density
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