Retinal explants were rinsed once in Dulbecco's PBS (Sigma-Aldrich Co. LLC) and fixed with 4% paraformaldehyde in PBS containing 4% sucrose, then incubated overnight in primary antibodies against YFP (rabbit anti-GFP; 1:2000; Abcam, Cambridge, MA or mouse anti-GFP; 1:1000; Millipore, Billerica, MA), phosphorylated neurofilaments (Smi-31; 1:1,000; Covance, Princeton, NJ), calretinin (1:1000; Chemicon International, Billerica, MA), or β-galactosidase (1:1000, Promega, Madison, WI), as indicated. Retinas were then labeled with species-specific secondary IgG antibodies conjugated to Alexa 488, 568, or 594 (Invitrogen), mounted in Hydromount medium (National Diagnostics, Atlanta, GA), and imaged using a Zeiss fluorescence microscope (Carl Zeiss Microimaging, LLC, Thornwood, NY) equipped with a charge-coupled device (CCD) camera (AxioCam MRm, Zeiss). Post-processing and quantification of images were done using ImageJ (National Institutes of Health) and Matlab algorithms, respectively.
Hydromount medium
Manufactured by National Diagnostics
Sourced in United States
Hydromount medium is a water-soluble mounting medium used for microscopy applications. It is designed to provide a stable environment for mounting and preserving specimens on microscope slides.
Automatically generated - may contain errors
Lab products found in correlation
Calretinin, by Merck Group (1 mentions)
Dulbecco s pbs, by Merck Group (1 mentions)
Axiocam mrm, by Zeiss (1 mentions)
Rabbit anti gfp, by Abcam (1 mentions)
Mouse anti gfp, by Merck Group (1 mentions)
Smi31, by Fortrea (1 mentions)
β galactosidase, by Promega (1 mentions)
Cm3050, by Leica (1 mentions)
Dapi nuclear stain, by Thermo Fisher Scientific (1 mentions)
Vibratome, by Leica (1 mentions)
Fv3000 confocal system, by Olympus (1 mentions)
Ab5392, by Abcam (1 mentions)
Pa1 933, by Thermo Fisher Scientific (1 mentions)
Goat anti chicken alexa 488, by Thermo Fisher Scientific (1 mentions)
Donkey anti mouse alexa 647, by Thermo Fisher Scientific (1 mentions)
Mab5406, by Merck Group (1 mentions)
D1306, by Thermo Fisher Scientific (1 mentions)
Sp8 confocal microscope, by Leica (1 mentions)
5 protocols using hydromount medium
1
Immunostaining Retinal Explants
2
Immunofluorescence Characterization of Spinal Cord Segments
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For the neurochemical characterization, L4–L5 segments were carefully dissected, post-fixed for 3 h in PFA and sectioned with a cryostat (Leica CM3050 S; Leica) at 100 µm thickness. Blocking was carried out with 5% normal donkey or goat serum (NDS/NGS; Sigma; St. Louis, MO, USA) for an hour followed by incubation in primary (2 days; 4 °C) and secondary (2hrs at room temperature, or overnight at 4 °C) antibody mixtures. All antibodies were diluted in Na-borohydride-containing phosphate buffer (0.1M PB) supplemented with a 0.3% Triton-X 100 and 1% (v/v) appropriate serum. Three 10-minute washes in 0.1 M PB were performed after incubation with all solutions except after the blocking step. Details of the primary antibodies applied in this study are presented in Table 4 . Species-specific secondary antibodies were raised in donkey or goat and conjugated to Alexa Fluor-488, 555 and 647 (Invitrogen). At the end of the protocol, cell nuclei-specific 4′,6-diamidino-2-phenylindole (DAPI) was added to help determine the laminar boundaries based upon the orientation and density of the nuclei. Sections were mounted in a Hydromount medium (National Diagnostics; Brandon, FL, USA) and confocal images were scanned with Olympus FV3000 confocal systems (Tokyo, Japan).
3
Immunofluorescent Staining of Organoids
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A standard protocol for immunofluorescent staining was followed. Briefly, organoids were fixed in 4% PFA for 1 h at RT. Wells were washed for 2.5 h with wash buffer (PBS with 1% donkey serum and 0.1% Triton X-100) on a rocker. Non-specific binding was blocked and organoids were permeabilised using PBS with 10% donkey serum and 1% Triton X-100 (blocking buffer) for 4 h at RT on a rocker. Primary rabbit (R) or mouse (M) antibodies (R anti-chromogranin A, R anti-mucin 2, R anti-e-cadherin and M anti-SAG1 (surface antigen 1)) were diluted 1:200 in blocking buffer; 200 μl was applied to each well and incubated at 4 °C overnight on a rocker followed by three washes with washing buffer. Donkey anti-rabbit FITC or TRITC or donkey anti-mouse Alexa Fluor (AF) 488 secondary antibodies, diluted 1:200 in wash buffer, with rhodamine or AF647-conjugated phalloidin (F-actin detection) diluted 1:250 were applied to the appropriate wells and incubated for a further 2 h at RT. Wells were washed with PBS for 2 h, with the addition of DAPI nuclear stain (Invitrogen, MA, USA) for the final 10 min. Coverslips were mounted onto slides with Hydromount medium (National Diagnostics, Nottingham, UK).
4
Immunofluorescent Labeling of Mouse Brain
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Immunofluorescent labeling was used for gaining the fluorescent green GFP signal and detecting the TRPC4, c-Fos, and BDNF in US-treated and control mouse brains. E18.5 embryos and P3 and P30 mouse pups were sacrificed by decapitation, and whole brains were carefully dissected and immersion fixed in 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS, pH 7.4) overnight at 4°C. Brain samples were then rinsed with PBS and embedded in 4% agarose, and 100-micron-thick free-floating coronal sections were made with a vibratome (Leica, Wetzlar, Germany). Sections were incubated in primary (2 days at 4°C) and secondary (overnight at 4°C) antibody mixtures. All antibodies were diluted in PBS (pH 7.4) supplemented with 0.3 M NaCl and 0.3% Triton X-100. Three 15-min washes in PBS were performed after incubation.
Details of the primary antibodies applied in this study are listed inTable 1 . Species-specific secondary antibodies were raised in donkey or goat and conjugated to Alexa Fluor-488, 555, and 647 (Invitrogen, Carlsbad, CA, United States). At the end of the protocol, the sections were incubated with cell nucleus-specific DAPI (Sigma, D9542, 100 ng/ml) for 2 h at RT to help determine layer boundaries. Sections were mounted in Hydromount medium (National Diagnostics Atlanta, GA, United States) and confocal images were obtained with an Olympus FV3000 confocal system.
Details of the primary antibodies applied in this study are listed in
5
Immunocytochemistry of Neuronal Markers
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For immunocytochemistry, neurons at DIV14 were washed with PBS once, then fixed with ice-cold 100% methanol for 10 minutes at -20°C. After three washes in PBS for 30 min at room temperature, cells were incubated with Chicken antibody against MAP2
(1:1000, ab5392, Abcam), Rabbit antibody against Parvalbumin (1:500, PA1-933, Thermo Fischer Scientific) and Mouse antibody against GAD67 (1:500, MAB5406, Sigma-Aldrich) in staining buffer (0.2% BSA, 0.8 M NaCl, 0.5% Triton X-100, 30 mM phosphate buffer, pH 7.4) overnight at 4°C. Neurons were then washed three times in PBS for 30 min at room temperature and incubated with Alexa Fluor conjugated secondary antibodies (Goat anti-Chicken-Alexa 488, A11039, Life Technologies; Goat anti-Rabbit-Alexa 568, A11011, and Donkey anti-Mouse-Alexa 647, A31571, Life Technologies) with 1:1000 dilution in staining buffer for 2 hours at room temperature and stained with DAPI (300 nM, D1306, Thermo Fisher Scientific) for 10 minutes at room temperature. After washed three times in PBS for 30 min, Cover glasses were mounted onto microscope slides with HydroMount medium (National Diagnostics).
Confocal images were acquired using a SP8 confocal microscope (Leica) with a 40x oil objective.
(1:1000, ab5392, Abcam), Rabbit antibody against Parvalbumin (1:500, PA1-933, Thermo Fischer Scientific) and Mouse antibody against GAD67 (1:500, MAB5406, Sigma-Aldrich) in staining buffer (0.2% BSA, 0.8 M NaCl, 0.5% Triton X-100, 30 mM phosphate buffer, pH 7.4) overnight at 4°C. Neurons were then washed three times in PBS for 30 min at room temperature and incubated with Alexa Fluor conjugated secondary antibodies (Goat anti-Chicken-Alexa 488, A11039, Life Technologies; Goat anti-Rabbit-Alexa 568, A11011, and Donkey anti-Mouse-Alexa 647, A31571, Life Technologies) with 1:1000 dilution in staining buffer for 2 hours at room temperature and stained with DAPI (300 nM, D1306, Thermo Fisher Scientific) for 10 minutes at room temperature. After washed three times in PBS for 30 min, Cover glasses were mounted onto microscope slides with HydroMount medium (National Diagnostics).
Confocal images were acquired using a SP8 confocal microscope (Leica) with a 40x oil objective.
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