Hippocampal neurons on coverslips were washed once in 1X artificial cerebrospinal fluid (ACSF) then fixed in a 4% paraformaldehyde/4% sucrose solution for 10 minutes. To immunostain the total protein, neurons were permeabilized with 0.3% Triton-X-100 (FisherBiotech) in phosphate buffered saline (PBS) for 8 minutes, followed with three rinses with 1X PBS. Neurons were then subjected to a one-hour blocking procedure in 10% goat-serum/PBS followed with another one-hour incubation with primary antibody at room temperature. After thoroughly rinsing to remove excess primary antibody, neurons were incubated with Alexa Fluor®-conjugated fluorescent secondary antibodies (1:700, Life Technologies) for another hour. Neurons were then mounted to microscopy glass slides with ProlongGold anti-fade mounting reagent (Life Technologies) and stored at 4°C until subsequent visualization.
Alexa fluor conjugated fluorescent secondary antibody
Manufactured by Thermo Fisher Scientific
Alexa Fluor-conjugated fluorescent secondary antibodies are a type of laboratory reagent used in various immunodetection techniques. These antibodies are designed to bind to primary antibodies, allowing for the visualization and detection of target proteins or other biomolecules in biological samples. The Alexa Fluor dyes provide bright fluorescent labeling, enabling sensitive and quantitative analysis.
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Lab products found in correlation
Prolong gold antifade mounting reagent, by Thermo Fisher Scientific (5 mentions)
Lsm 710 microscope, by Zeiss (1 mentions)
Zen software, by Zeiss (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Triton x 100 tx 100, by Merck Group (1 mentions)
Dragonfly spinning disk, by Oxford Instruments (1 mentions)
Glass bottomed dishes, by MatTek (1 mentions)
9 protocols using alexa fluor conjugated fluorescent secondary antibody
1
Immunostaining of Hippocampal Neurons
2
Immunofluorescence Staining of Cultured Neurons
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Culture neurons were washed twice in ice-cold PBS and fixed in a 4% paraformaldehyde solution at room temperature for 10 min. Cell membranes were then permeabilized for 8 min in 0.3% Triton X-100 (Fisher Biotec; cat. #BP151-100) in PBS. Cells were rinsed three times with PBS, and blocked with 10% goat serum for 1 h. Primary antibodies (in 5% goat serum PBS) were applied overnight at 4 °C, followed by washing twice with PBS and incubated with the appropriate Alexa Fluor-conjugated fluorescent secondary antibodies (1:400, Life Technologies) for an additional hour. Cells were then rinsed (with Hoechst 1:2000 in the first rinse) and mounted on microscopy glass slides using Prolong Gold anti-fade mounting reagent (Invitrogen; cat. #P36930) and stored in the dark at 4 °C for subsequent imaging.
3
Immunofluorescence Staining of Cultured Neurons
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Culture neurons were washed twice in ice-cold PBS and fixed for 10 min in a 4% paraformaldehyde solution at room temperature. Cell membranes were then permeabilized for 8 min in 0.3% Triton X-100 (Fisher Biotec; cat. #BP151–100) in PBS. Cells were rinsed three times in PBS, and blocked in 10% goat serum for 1 h. Primary antibodies (in 5% goat serum PBS) were added, and the cells were incubated overnight at 4℃, washed twice with PBS and incubated with appropriate Alexa Fluor-conjugated fluorescent secondary antibodies (1:400, Life Technologies) for an additional hour. Cells were then rinsed (with Hoechst 1:2000 in the first rinse) and mounted to microscopy glass slides with Prolong Gold anti-fade mounting reagent (Invitrogen; cat. #P36930) and stored at 4℃ in dark for subsequent imaging.
4
Immunostaining of Hippocampal Neurons
5
Immunocytochemistry of Hippocampal Neurons
6
Caco-2/TC7 Cell Imaging Protocol
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Caco-2/TC7 cells were fixed with 4% paraformaldehyde (PFA), permeabilized by 0.05% saponin or 0.01% digitonin (for LC3 staining) in phosphate-buffered saline (PBS)/10% serum, incubated with primary antibodies in the same buffer, and then rinsed in PBS and incubated with appropriate cyanin dyes or Alexa Fluor–conjugated fluorescent secondary antibodies (Invitrogen). When indicated, cells were stained for neutral lipids with BODIPY 493/503 (10 μg/ml). For the labeling of PI3P with FYVE-FYVEGST, cells were fixed and incubated for 1 h with purified FYVE-FYVEGST recombinant protein (20 μg/ml final concentration), washed with PBS, and labeled with a FITC-conjugated anti-GST antibody. After nuclear staining by DAPI and postfixation with 4% PFA, samples were examined by laser scanning confocal microscopy (LSM 710 microscope; Carl Zeiss). All confocal acquisitions, at subapical, nuclear, or basal plans, were analyzed (lipid droplet diameter, 3D reconstructions, XZ projections, etc.) with ZEN software (Carl Zeiss). Lipid droplets and/or number of autophagosome/endosome structures were manually counted in confocal sections (1000 μm2). The value of total lipid droplet content used throughout this article corresponds to n (number of lipid droplets/1000-μm2 cell field) × v (mean volume, in μm3, of lipid droplets in the same area) and is expressed as arbitrary units.
7
Immunofluorescence Staining of Organoids and UE
8
HCN2 Channel Immunodetection in Embryos
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HCN2 channel detection was performed by immunofluorescence for HCN2 channel. Briefly, embryos were fixed overnight in MEMFA at 4 °C (ref. 71 ). The embryos were permeabilized in PBS 0.1% Triton X-100; blocked with 10% goat serum in PBST for 1 hour at room temperature; and incubated at 4 °C overnight with primary antibody (Anti-HCN2: ThermoFisher Scientific PA1-918) at 1:500 dilution in PBST + 10% goat serum (blocking buffer). Embryos were washed six times in PBST and incubated with Alexa Fluor-conjugated fluorescent secondary antibody (Invitrogen) at 1:500 dilution in PBST + 10% goat serum overnight at 4 °C. Sections were washed six times and photographed using an Olympus BX-61 microscope equipped with a Hamamatse ORCA AG CCD camera, and controlled by MetaMorph software. NIH ImageJ software was used to quantify the fluorescence intensities of the immunostained embryos.
9
Embryonic Expression Patterns of HCN4
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Spatial distribution of HCN4 channel in embryos was detected by immunofluorescence for the HCN4 channel on whole embryos. Briefly, embryos were fixed overnight in MEMFA at 4°C (Sive et al., 2000 ). The embryos were permeabilized in PBS 0.1%Triton-X-100, blocked with 10% goat serum in PBST for 1 h at room temperature, and incubated at 4°C overnight with primary antibody (Anti-HCN4 – rabbit polyclonal; Abcam ab66501) for HCN4 at 1:500 dilution in PBST+10% goat serum (blocking buffer). Embryos were washed six times in PBST and incubated with Alexa Fluor-conjugated fluorescent secondary antibody (Invitrogen) at 1:500 dilution in PBST+10% goat serum overnight at 4°C. Embryos were washed six times in PBST and photographed using Nikon SMZ-1500 scope with Q-capture software.
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