HaCaT and NCI-H23 cells were cultured on glass coverslips, fixed with 4% paraformaldehyde-PBS, permeabilized with 0.3% Triton X-100/PBS, and incubated with 1% BSA. Cells were then incubated with anti-TMEPAI 9F10 antibody and then with Alexa 488-labeled goat anti-mouse IgG (Molecular Probes, Eugene, OR, USA). The nuclei were stained with Hoechst 33342 (Sigma). Intracellular localization was then observed by fluorescence microscopy (Axiovert 200; Zeiss, Oberkochen, Germany).
Alexa 488 labeled goat anti mouse igg
Manufactured by Thermo Fisher Scientific
Sourced in United States
Alexa 488-labeled goat anti-mouse IgG is a fluorescent secondary antibody used for the detection of mouse primary antibodies in various immunological applications. It is conjugated with the Alexa Fluor 488 dye, which provides bright green fluorescence upon excitation.
Automatically generated - may contain errors
Lab products found in correlation
Hoechst 33342, by Merck Group (3 mentions)
Axiovert 200, by Zeiss (2 mentions)
Fluoview fv1000d, by Olympus (1 mentions)
Mouse anti gfp antibody, by Merck Group (1 mentions)
Fluoromount, by Diagnostic BioSystems (1 mentions)
Anti c myc n 262, by Santa Cruz Biotechnology (1 mentions)
Texas red labeled goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 nhs, by Thermo Fisher Scientific (1 mentions)
Anti cytochrome c antibody, by Cell Signaling Technology (1 mentions)
Ab104139, by Abcam (1 mentions)
Fv1000, by Olympus (1 mentions)
Ab199, by Abcam (1 mentions)
Sc 659, by Santa Cruz Biotechnology (1 mentions)
Cell tak, by BD (1 mentions)
Alexa594 labeled goat anti rabbit, by Thermo Fisher Scientific (1 mentions)
Prolong glass antifade mountant with nucblue stain, by Thermo Fisher Scientific (1 mentions)
Axio observer, by Zeiss (1 mentions)
Bz x700 fluorescence microscope, by Keyence (1 mentions)
Transwell plate, by Corning (1 mentions)
12 well tissue culture plate, by Corning (1 mentions)
14 protocols using alexa 488 labeled goat anti mouse igg
1
Immunocytochemical Localization of TMEPAI
2
Colocalization of CD133, 8-Nitroguanine, and 8-Oxodg
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Double or single fluorescent immunohistochemistry was performed to examine the colocalization of CD133, 8-nitroguanine, and 8-oxodG as described previously [25 (link)]. Paraffin sections were incubated with the primary antibodies [rabbit polyclonal anti-CD133 antibody (1 : 500, Abcam, Cambridge, UK), rabbit polyclonal anti-8-nitroguanine antibody (1 μg/mL) produced by our group [25 (link), 26 (link)], and mouse monoclonal anti-8-oxodG antibody (1 : 200, Japan Institute for the Control of Aging, Fukuroi, Japan)] overnight at room temperature. The sections were next incubated with fluorescent secondary antibodies (Alexa 488-labeled goat anti-mouse IgG and/or Alexa 594-labeled goat anti-rabbit IgG antibodies, 1 : 400 each, Molecular Probes Inc., Eugene, Oregon, USA) for 3 h at room temperature. Finally, the nuclei were stained by 4′-6-diamidino-2-phenylindole (DAPI) and the sections were examined with a fluorescence microscope (LX70, Olympus, Tokyo, Japan) or a laser scanning confocal microscope (Fluoview FV1000-D, Olympus) [10 (link)].
3
Immunohistochemical Analysis of GCaMP6s Expression
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Following the recording sessions, the monkeys were deeply anesthetized with an intravenous injection of sodium pentobarbital (70 mg/kg, i.v.) and transcardially perfused with 0.01 M PBS followed by 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). Brains were extracted and post-fixed in 4% paraformaldehyde overnight at 4 °C and cryoprotected with increasing gradients of sucrose (5%, 10%, and 20%). Frozen brains were then sliced into coronal sections at 40-µm thickness using a cryostat.
One in four successive sections was immunohistochemically stained. Free-floating sections were washed with PBS and permeabilized in PBS containing 0.3% Triton X-100 (PBST). After blocking for 1 h in 3% normal goat serum in PBST containing 1% bovine serum albumin (BSA-PBST), sections were incubated for 2 nights at 4 °C with a mouse anti-GFP antibody (1:250, Millipore, MA, USA) in BSA-PBST. After washing in PBST, sections were incubated for 4 h at 20 °C with Alexa-488 labeled goat anti-mouse IgG (1:1000, Molecular Probes, OR, USA) in BSA-PBST. After washing in PBS, the sections were mounted on glass slides with Fluoromount (Diagnostic BioSystems, CA, USA). GCaMP6s fluorescence images were acquired using a camera lucida attached to an epifluorescence microscope (BX51, Olympus, Tokyo, Japan) with 10 × , 20 × , and 40 × objective lenses.
One in four successive sections was immunohistochemically stained. Free-floating sections were washed with PBS and permeabilized in PBS containing 0.3% Triton X-100 (PBST). After blocking for 1 h in 3% normal goat serum in PBST containing 1% bovine serum albumin (BSA-PBST), sections were incubated for 2 nights at 4 °C with a mouse anti-GFP antibody (1:250, Millipore, MA, USA) in BSA-PBST. After washing in PBST, sections were incubated for 4 h at 20 °C with Alexa-488 labeled goat anti-mouse IgG (1:1000, Molecular Probes, OR, USA) in BSA-PBST. After washing in PBS, the sections were mounted on glass slides with Fluoromount (Diagnostic BioSystems, CA, USA). GCaMP6s fluorescence images were acquired using a camera lucida attached to an epifluorescence microscope (BX51, Olympus, Tokyo, Japan) with 10 × , 20 × , and 40 × objective lenses.
4
Immunofluorescence Analysis of TSC-22 and c-MYC
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Immunofluorescence in HaCaT cells stably expressing TSC‐22 was carried out using anti‐TSC‐22 and anti‐c‐MYC (N262; Santa Cruz) primary antibodies followed by incubation with Alexa 488‐labeled goat anti‐mouse IgG and Texas Red‐labeled goat anti‐rabbit IgG secondaries (Molecular Probes, Eugene, OR, USA). Nuclei were stained with Hoechst 33342 (Sigma). Intracellular localization of TSC‐22 and c‐MYC was observed using a fluorescence microscope (Axiovert 200; Carl Zeiss AG, Oberkochen, Germany).
5
Immunohistochemistry of C. elegans Neurotransmitter Receptors
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The N2, frm-3(gk585) or lin-2(n397) animals were grown and collected from NGM rich medium plates. A freeze/cracking step was performed before acetone/methanol fixation at −20 °C. Primary antibody rabbit anti-UNC-49 was diluted at 1:250; mouse anti-UNC-17 (VAChT) was diluted at 1:50079 (link). Secondary antibodies Cy3-labeled goat anti-rabbit IgG was diluted at 1:500 (Molecular Probes, A10520), Alexa 488-labeled goat anti-mouse IgG ((Molecular Probes, A-11001) was diluted at 1:500.
6
Quantifying UVB-Induced DNA Lesions
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To evaluate the capacity of UVB radiation to induce the development of DNA lesions, thymine dimers formation was quantified by flow cytometry. After UVB radiation with a total dose of 0.25 J/cm2, HaCat cells, RHE, or fish embryos were incubated 10 min. After that, HaCat cells, RHE, and embryo cells were disaggregated with trypsin at 37 °C and fixed with 4% paraformaldehyde (PFA) (Electron Microscopy Sciences, Madrid, Spain). Following, they were incubated with primary antibody mouse monoclonal anti-Thymine Dimer (H3) (Abcam, Cambridge, UK) and stained with secondary antibody Alexa 488 labeled goat anti-mouse IgG (Invitrogen, Fisher Scientific, Madrid, Spain). Finally, samples were analyzed by BD Flow Cytometer (BD Biosciences, San Jose, CA, USA).
7
Characterization of Virus-like Particles
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For evaluation of estimated molecular masses of the particles, the supernatants of cells transfected with prME-expressing plasmids were reacted with Alexa Fluor 488-NHS (Invitrogen); free dye subsequently was removed using a gel filtration column. For analysis of epitope on the surfaces of the particles, the supernatants were incubated with SLT-8 antibody or normal mouse IgG for 30 min at room temperature, followed by incubation with Alexa 488-labeled goat anti-mouse IgG (Invitrogen) for 30 min at room temperature. A commercial FCS setup (FCS-101B, Hamamatsu Photonics, Shizuoka, Japan), which is equipped with a water-immersion objective (UPAPLO 40 × /1.2 W, Olympus), was used. The 473-nm line of a semiconductor laser (output power 1 mW) was applied and attenuated adequately with light filters. The laser beam was focused at about 0.2 mm above the bottom of cover glasses in a typical sample droplet volume of 20 μL. Fluorescence intensity data through an optical filter (>500 nm) located adjacent to the photo multiplier tube were collected. Autocorrelation analysis of the fluorescence intensity fluctuations was performed using the control software supplied by the manufacturer.
8
Immunofluorescent Detection of Cytochrome c
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After staining the cells with MTR-CMX cells were washed with PBS, fixed with 3.7% paraformaldehyde in PBS for 15 min and permeabilized with 0.25% (v/v) Triton X-100/PBS for 15 min at RT. Following permeabilization, cells were washed twice with PBS and nonspecific absorption was blocked using UltraVision blocking reagent (Thermo Fisher Scientific) for 10 min at RT. Then, cells were incubated at 4 °C overnight with anti-Cytochrome c antibody (Cell Signaling, # 12963; 1:100 in Antibody Diluent, Dako). After washing with PBS, Alexa488-labeled goat-anti-mouse IgG (Invitrogen, # A11001; 1:300 in Antibody Diluent) was used as secondary antibody (60 min, RT). Slides were stored in 1% (w/v) BSA/PBS until further use.
9
Immunofluorescence Analysis of Blastocysts
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Blastocysts were fixed in 3.7% paraformaldehyde in PBS for 20 min at room temperature and washed twice in PBS containing BSA (BPBS). Embryos were permeabilized with 0.2% Triton X-100 in PBS for 30 min, then washed twice in BPBS. Embryos were incubated in blocking solution (0.1% Tween 20 in BPBS) for 1 h and then incubated with primary antibodies against HIF-2α (Abcam, USA; ab199) (1:100), MnSOD (Abcam; ab16956) (1:200), and LIFR (Santa Cruz, USA; sc-659) (1:200) overnight at 4 °C. On the following morning, embryos were rinsed three times in BPBS and incubated in the appropriate secondary antibody conjugated with Alexa 488–labeled goat antimouse IgG (Invitrogen; A-11029) (1:100) or Alexa 568–labeled goat antimouse IgG (Invitrogen; A-11036) (1:100) for 1 h in the dark. After washing in PBS, the stained embryos were mounted in Fluoroshield mounting medium with 4′,6-diamidino-2-phenylindole (DAPI; Abcam, ab104139) and observed by confocal laser-scanning microscopy (FV1000; Olympus, Japan) to detect the fluorescence. For image analysis, the intensities of green fluorescence (Alexa 488) and red fluorescence (Alexa 568) were measured using Olympus Fluoview® software (FV10-ASW).
10
Immunostaining of β-Catenin and Pericentrin
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Cells were grown on coverslips coated by Cell-Tak (BD Biosciences), washed with PBS and fixed for 20 min in 4% PFA. Cells were then permeabilized in 0.5% Triton X-100 for 3 min. After blocking, cells were immunostained for 1 hr with a primary antibody, and after subsequent washes the cells were incubated for 1 hr with secondary fluorescent antibodies. Primary antibodies: mouse anti-β-catenin and rabbit anti-pericentrin (Abcam, cat# ab4448). Secondary antibodies: Alexa488-labeled goat anti-mouse IgG and Alexa594-labeled goat anti-rabbit (Invitrogen, Carlsbad, CA). Nuclei were counterstained with Hoechst 33342 (Sigma) and coverslips were mounted in mounting medium.
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