Sequential immunofluorescence (IF) and smFISH on fixed myotubes was performed. Briefly, C2C12 myotubes were differentiated for 7 days in differentiation media, fixed in 4% paraformaldehyde (4%) for 10 minutes, and washed in PBS. The following antibodies were used for immunofluorescence: rabbit anti-TDP-43 (Proteintech, 1:400), rabbit anti-A11 oligomer (Thermo Fisher Scientific, 1:400), goat anti-rabbit Alexa 647 (Abcam, 1:1000), goat anti-mouse IgG1 Alexa 488 (Thermo Fisher Scientific, 1:1000). All IFs were performed sequentially except for staining with mouse anti-myosin heavy chain, F59 (DSHB) which was diluted (1:10) in hybridization buffer. Custom Stellaris FISH probes were designed against mouse Titin, Myosin-3, Troponin C1 and labeled with Quasar 570 Dye using Stellaris RNA FISH Probe Designer (Biosearch Technologies, Petaluma, CA).
Goat anti mouse igg1 alexa 488
Manufactured by Thermo Fisher Scientific
Sourced in United States
Goat anti-mouse IgG1 Alexa 488 is a secondary antibody that binds to mouse IgG1 antibodies. It is labeled with the Alexa Fluor 488 fluorescent dye, which emits green fluorescence when excited by a suitable light source.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 710 confocal microscope, by Zeiss (3 mentions)
Rabbit anti a11 oligomer, by Thermo Fisher Scientific (2 mentions)
Goat anti rabbit alexa 647, by Abcam (2 mentions)
Rabbit anti tdp 43, by Proteintech (2 mentions)
Stellaris rna fish probe designer, by Biosearch Technologies (2 mentions)
Goat anti mouse igg2b alexa 647, by Thermo Fisher Scientific (2 mentions)
Ab16669, by Abcam (1 mentions)
Ab64088, by Abcam (1 mentions)
Ab10558, by Abcam (1 mentions)
Ab31630, by Abcam (1 mentions)
Ab64693, by Abcam (1 mentions)
Alexa 594 goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Ab15323, by Abcam (1 mentions)
Mouse anti huc d, by Thermo Fisher Scientific (1 mentions)
Prolong gold antifade mountant with dapi, by Thermo Fisher Scientific (1 mentions)
Rabbit anti neun, by Merck Group (1 mentions)
Rat anti f4 80, by Bio-Rad (1 mentions)
Rat anti mbp, by Bio-Rad (1 mentions)
Rabbit anti s100, by Agilent Technologies (1 mentions)
Rabbit anti ng2, by Merck Group (1 mentions)
12 protocols using goat anti mouse igg1 alexa 488
1
Multimodal Analysis of Myotube Transcripts
2
Multimodal Analysis of Myotube Transcripts
3
Immune Response to Biomaterials in Rats
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Nine male Sprague‒Dawley rats (280–300 g) were used to evaluate the immune response and cell infiltration of different materials. 1 mL mECM hydrogel, PM (3 mg) in 1 mL saline and 1 mL mECM + PM composites were prepared under sterile conditions and loaded into a 2.5 mL syringe, respectively. Then, they were subcutaneously injected into rats for 1 and 4 weeks. H&E staining was used to assess cell infiltration. Masson staining was used to assess the collagen deposition and distribution. To evaluate the immune response, immunofluorescence staining was performed to identify the immune cells with the following antibodies: CD68 (1:250, Abcam, ab31630), iNOS (1:300, Abcam, ab15323), CD206 (1:300, Abcam, ab64693), CD45 (1:150, Abcam, ab10558), CD20 (1:100, Abcam, ab64088) and CD3 (1:100, Abcam, ab16669). Subsequently, the following secondary antibodies were reacted with the corresponding primary antibodies: goat anti-rabbit IgG Alexa 594 (1:500, Invitrogen, USA) and goat anti-mouse IgG1 Alexa 488 (1:500, Invitrogen, USA). DAPI staining was used to label the cell nucleus (Southern Biotech, England). Finally, images were observed using a confocal laser microscope (Leica, SP8, Germany). The S/L value of microspheres, cell infiltration, and fluorescence intensity of iNOS and CD206 were measured using Image-Pro Plus 6.0 software.
4
Quantifying Neuronal Proliferation in Zebrafish
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To assay neuronal proliferation, we labeled with anti-HuC/D (Elav protein) (Invitrogen A21271). Control (0.08% DMSO) and LY411575 groups (100 nM and 8 μM) were prepared as described above. At 24 hpf, embryos were fixed overnight using 4% paraformaldehyde in 1× PBS at 4°C. Washes were performed with 1× PBS containing 0.1% TritonX-100. We used primary antibody mouse anti-HuC/D (1:500, Invitrogen, 16A11). Secondary detection was performed with goat anti-mouse IgG1 Alexa 488 (1:500, Invitrogen, A32723). To analyze images, signal intensity of a 56 μm × 6 μm (W × H) region spanning a lateral to midline hemi-section of the anterior spinal cord was recorded using ImageJ. Three sections were measured per larva, averaged and standardized for comparison between groups.
5
Immunofluorescent Staining of IL-20/22 Receptors
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OCs were grown from RA SFMCs on sterile glass slides in 24-well cell culture plates and stained for confocal microscopy as previously described [35 (link)]. Briefly, cells were fixed with 4 % paraformaldehyde for 10 minutes at RT. Non-specific binding was blocked by incubating in PBS with 0.5 % BSA and 5 % goat serum for 30 minutes at RT. Cells were stained with either anti-IL-20R1 IgG1 (173714; R&D Systems) or anti-IL-22R1 IgG1 (305405; R&D Systems) in combination with goat anti-mouse IgG1 Alexa 488 (Invitrogen). Cells were co-stained with anti-TRAP IgG2b in combination with goat anti-mouse IgG2b Alexa 647 (Invitrogen). Isotypes served as negative controls. Glass slides were placed in Prolong Gold Antifade Mountant with DAPI (Life Technologies) and allowed to dry overnight. All micrographs were collected using a Zeiss LSM-710 confocal microscope.
6
Immunolabeling of Myelinating Cultures
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Post-fixation, myelinating cultures were permeabilised in ethanol (−20 °C; 10 min) and incubated in primary antibodies in 10% goat serum overnight at 4 °C. Mouse anti-ZIKV [clone 0302156 Aalto Bio; 1 in 500] was used in combination with one or other of the following cell-type specific antibodies: rat anti-PLP/DM20 [Clone AA3, kindly provided by Dr. Steven Pfeiffer, Connecticut; 1 in 400]; rabbit anti-NeuN [Millipore; 1 in 750], rabbit anti-S100 [Dako; 1 in 400], rabbit anti-GFAP [Dako; 1 in 1000], rabbit anti-NG2 [Millipore; 1 in 200], rat anti-MBP [AbD serotec; 1 in 500], or rat anti-F480 [AbD serotec; 1in 600]). rat anti-MBP was also used in combination with mouse SMI31 anti-phosphorylated heavy and medium chain neurofilament [Biolegend; 1 in 1500] and rat anti-F480 in combination with mouse SMI32 anti-non-phosphorylated neurofilament heavy chain [Sternberger; 1 in 1500] or mouse anti-β-Tubulin III [Sigma; 1 in 200]. After washing, secondary antibodies (goat anti-mouse IgG1 Alexa 488 and goat anti-rat IgG or goat anti-rabbit IgG or goat anti-mouse IgG2a Alexa 594; Invitrogen) were applied for 1 h at room temperature. Coverslips were mounted on glass slides in Citifluor mounting medium with DAPI (1 ng/ml; Electron Microscopy Sciences Pennsylvania US) and sealed with nail enamel.
7
Cardiac Aggregates Dissociation and Characterization
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Cardiac aggregates were dissociated with 0.5% trypsin followed by fixation and permeabilization using Cytofix/Cytoperm reagent (BD Biosciences) according to the manufacturer. Cell were then stained with primary mouse anti-cTnT (Abcam, 1:500) overnight followed by cardiomyocytes staining with the secondary antibody, goat anti-mouse IgG1 Alexa488 (Life Technologies, 1:500). After 1h of secondary antibody incubation, the cells were washed and assayed on a flow cytometer.
8
Isolation and Culture of Satellite Cells
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Satellite cells were isolated from the hind leg muscles of approximately 20 to 25-week-old wildtype and miR-206/133b knock-out animals by using the skeletal muscle dissociation kit (Miltenyi Biotech, Bergisch Gladbach, Germany, 130-098-305) and enriched by the satellite cell isolation Kit, mouse (Miltenyi Biotech, 130-104-268) according to the manufacturer’s instructions. Isolated cells were plated on gelatin-coated μclear 96-well plates (Sigma#M0562). Satellite cells were grown in proliferation medium (40% DMEM, 40% Ham F-10; 20% FCS; Pen/Strep; 2.5 ng/ml human FGF-2, Miltenyi Biotech#130-093-840) for 3 days, followed by switch to differentiation medium (DMEM, 2% horse serum, and Pen/Strep). Cells were incubated for 5 min in fixative (4% PFA/PBS, 0.1% sodium desoxycholate, 0.2% NP-40), washed 3 times in PBS, blocked in carrier (PBS, 5% BSA, 0.5% NP-40) for 1 h and then incubated with MF 20 supernatant in carrier (1:100, DSHB, Iowa City, Iowa). Secondary antibody was goat anti-mouse IgG1 Alexa 488 (1:2000, Life technologies).
9
Immunofluorescent Staining of Cardiomyocytes
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In preparation for immunofluorescent staining, plated cardiomyocytes were washed once with PBS. The cells were fixed and permeabilized by applying Cytofix/Cytoperm für 20 minutes. After fixation, the cells were washed with Permwash (BD Biosciences) and stained with primary antibody dilutions for rabbit anti-cardiac troponin T (Abcam, 1:200) mouse anti-α-actinin (Abcam, 1:1000), rabbit anti-myosin light chain 2 (MLC2v) (Proteintech, 1:200), rabbit anti-connexin 43 (Abcam, 1:1000) and rabbit anti-N cadherin (Abcam, 1:200) overnight. The next day, the cells were washed and stained with the respective secondary antibodies anti-rabbit IgG Alexa594, goat anti-mouse IgG1 Alexa488, donkey anti-rabbit IgG Alexa488 or goat anti-mouse IgG1 Alexa594 (all Life Technologies, 1:1000). Next, the stained cardiomyocytes were washed, stained with DAPI to visualize nuclei and evaluated with a Zeiss Observer Z1 microscope.
10
Visualizing Chondroitin Sulfate Proteoglycans
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U373 astrocytes were incubated overnight with antibodies directed against chondroitin sulfate proteoglycans (CSPGs). Intact chondroitin sulfate side‐chains were detected with the 2H6 antibody (1:200, clone 2H6, Cosmo Bio, Germany) and core proteins using the BE‐123 antibody (1:200, clone BE‐123, Millipore, Germany,) following chABC predigestion. Subsequently, samples were incubated for 1 hr with Alexa‐488‐conjugated secondary antibodies (goat antimouse IgG1‐Alexa 488, 1:200; Molecular Probes, USA). Coverslips were mounted in MOWIOL after washing. Fluorescence analysis was performed with a Leica DM6000 microscope (Leica Microsystems, Germany).
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