Retinal whole mounts with fills of OFF-α T RGCs were fixed in 4% paraformaldehyde (w/v) solution for 30 min, washed in phosphate-buffered saline (PBS) (pH 7.4) for 1 hour, and subsequently placed into 12-well clear multiwell plates (Corning Falcon) for processing through free-floating immunochemistry. Retinas were then blocked with 5% normal donkey serum (NDS) in 0.3% PBTX (10× PBS + Triton X-100) solution for 2 hours and then incubated with Alexa Fluor 488–conjugated streptavidin (1:200; Invitrogen), mouse anti-AnkyrinG (1:200; NeuroMab), rabbit anti-Nav1.6 (1:200; MilliporeSigma), and goat anti–choline acetyl-transferase (ChAT) (1:200; MilliporeSigma) in 1% NDS with 0.3% PBTX solution for five overnights. Following incubation in primaries, samples were washed in PBS for 2 hours (4× for 30 min) and placed in Alexa Fluor 488–conjugated streptavidin (1:200; Invitrogen), Cy5-conjugated donkey anti-mouse (1:200; Jackson ImmunoResearch), Alexa Fluor 405–conjugated donkey anti-rabbit (1:200; Abcam), and Alexa Fluor 594–conjugated donkey anti-goat (1:200; Invitrogen) secondary antibodies in 0.3% PBTX solution for one overnight. Last, samples were washed in PBS for 1 hour (2× for 30 min), mounted with ProLong Gold antifade mounting medium (Invitrogen), coverslipped, and placed in 4°C refrigeration until further confocal imaging.
Prolong gold antifade mounting medium
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ProLong Gold antifade mounting medium is a reagent used in fluorescence microscopy to preserve and protect fluorescent signals in fixed and stained samples. It is designed to retard photobleaching and maintain the integrity of fluorescent proteins and dyes.
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Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (21 mentions)
Triton x 100, by Merck Group (15 mentions)
Hoechst 33342, by Thermo Fisher Scientific (10 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (9 mentions)
Paraformaldehyde (pfa), by Merck Group (6 mentions)
Bovine serum albumin (bsa), by Merck Group (6 mentions)
Lsm 710 confocal microscope, by Zeiss (5 mentions)
Tcs sp5 aobs laser scanning confocal microscope, by Leica (5 mentions)
Alexa fluor 488 goat anti rabbit igg, by Thermo Fisher Scientific (4 mentions)
Normal donkey serum (nds), by Jackson ImmunoResearch (4 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (4 mentions)
Lsm 880 confocal microscope, by Zeiss (4 mentions)
Paraformaldehyde (pfa), by Electron Microscopy Sciences (4 mentions)
Hoechst 33342, by Merck Group (4 mentions)
Lsm 700, by Zeiss (4 mentions)
Hoechst 33258, by Thermo Fisher Scientific (3 mentions)
A1si spectral detector confocal system, by Nikon (3 mentions)
Deadend fluorometric tunel system, by Promega (3 mentions)
Lsm 780, by Zeiss (3 mentions)
Lsm 780 confocal microscope, by Zeiss (3 mentions)
236 protocols using prolong gold antifade mounting medium
1
Retinal Whole Mount Immunostaining
2
Quantifying Mammalian Ovarian Germ Cells
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Ovaries isolated at 18.5 or 19.5 dpc were individually transferred into 1.5 ml low-retention microfuge tubes and dissociated into single cells as previously described23 (link),24 (link). The cell suspension was incubated in a hypotonic solution (0.45 % NaCl, pH 8.0) within a QuadriPERM 4-well chamber on histology slide (Lab-Tek II 154917) for 10 min and spun down, followed by fixation and washing. The slides were vacuum dried and stored in sealed boxes with silica gel at −20 °C. The microspread ovarian cells on histology slides were incubated with the primary antibodies overnight, the secondary antibodies for 1 h, and avidin-FITC for 1 h all at room temperature. (Details of the primary and secondary antibodies are given in Supplementary Tables S2 and S3 .) After IF-staining, the slides were washed, air-dried and mounted in the Prolong Gold Antifade mounting medium containing DAPI (Invitrogen P36935). The total number of germ cells was counted for each ovary using a Leica DM6000B microscope (Germany) at ×20 magnification. Around 50 cells/sample were analyzed for meiotic substages according to the IF-staining of SCP3, CREST, and γH2AFX. Localization and pattern of γH2AFX staining was also recorded. Co-localization of RAD51 was performed by omitting TRA98 while pachytene oocytes were identified by synapsis of SCP3-positive SC cores.
3
Visualizing Osteoclast Cytoskeletal Dynamics
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BMMs were cultured with 30 ng/ml M-CSF in the absence or presence of 0.4 μg/mL NGA for 2 hours. After a 30 minute stimulation with 50 ng/ml RANKL, the cells were fixed with 4% paraformaldehyde for 20 minutes, permeated with 0.1% Triton X-100 for 10 minutes, and blocked with 1% bovine serum albumin for 1 hour. The cells were then stained with the red-orange fluorophore, rhodamine phalloidin (Invitrogen) for at least 1 hour at 4°C, washed with PBS, and mounted using Prolong Gold antifade mounting medium (Invitrogen) for confocal microscopy.
4
Fluorescent Nuclei Imaging Protocol
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Immunolabeled cells were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) provided in ProLong Gold antifade mounting medium (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) to visualize nuclear morphology. Digital images were acquired at the Korea Basic Science Institute Gwangju Center using a TCS SP5 AOBS laser-scanning confocal microscope (Leica Microsystems, Heidelberg, Germany).
5
Immunofluorescent Visualization of Androgen Receptors
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AR immunoreactivity was visualized using a modified tyramide signal amplification method as described previously.42 Brain sections were rinsed with 0.1 M PBS, incubated in 0.6% hydrogen peroxide for 30 min, rinsed with 0.1 M PBS and then blocked with 3% normal donkey serum with 0.25% TritonX‐100 for 1 h at room temperature. Sections were incubated overnight with rabbit anti‐AR antibody (dilution 1:200; AbCam [EPR1535(2)], catalog. no. ab133273; RRID: AB_11156085). A series with no primary antibody was included as a negative control (Figure 1A,B ). Sections were rinsed with 0.1 M PBS and then incubated for 1 h with biotinylated donkey anti‐rabbit immunoglobulin G (dilution 1:500; Jackson ImmunoResearch Laboratories, catalog. no. 711‐065‐152; RRID: AB_2340593), followed by incubation in avidin‐biotin solution in 0.1 M PBS (dilution 1:1000; Vector Laboratories) for 1 h. Next, sections were incubated in biotinylated tyramide (dilution 1:250; Perkin Elmer) with 0.009% hydrogen peroxide for 10 min, followed by incubation with streptavidin‐conjugated AlexaFluor 594 (dilution 1:1000; Invitrogen, ThermoFisher) for 1 h. Sections were mounted onto gelatin‐coated slides and coverslipped with ProLong Gold Antifade mounting medium (Invitrogen, ThermoFisher).
6
Visualizing MRGPRX2 Receptor Trafficking
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Receptor trafficking after SP stimulation was modified from previously described antibody feeding assay [42 (link)]. RBL-2H3 cells expressing either WT-MRGPRX2 or its variants were plated onto sterilized glass coverslips (2 × 105 cells/12 mm diameter coverslip in a 24-well plate) and incubated overnight at 37 °C with 5% CO2 to allow attachment. Cells were rinsed with PBS and blocked with blocking buffer (PBS with 2% BSA) for 30 min at room temperature. Primary antibody incubation was performed using purified anti-MRGPRX2 antibody (1:250 dilution) for 1 h at 4 °C to label the cell surface expressed receptors. Cells were then stimulated with SP for 30 min at 37 °C to allow internalization, followed by being fixed with 4% paraformaldehyde for 15 min at 4 °C. Labeled surface receptors were detected by incubating with saturated Alexa Fluor 647-conjugated secondary antibody (red) for 1 h at 4 °C. Cells were then permeabilized using 0.2% Triton X-100 in blocking buffer for 30 min and internalized receptors were detected by incubating with Alexa Fluor 488-conjugated secondary antibody (green) for 30 min at 4°C. Then, cells were mounted onto the glass slides using ProLong Gold Antifade mounting medium (Invitrogen) and images were visualized using a Nikon Eclipse Ni microscope.
7
Visualizing CSF Circulation in Mice
8
Immunofluorescence Imaging of Endosomal Markers
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Fibroblasts were grown on coverslips, serum‐starved for 2 h—for consistency with transferrin feeding experiments—and fixed with 4% paraformaldehyde, 4% sucrose in 0.1 M PBS for 15 min, washed three times with PBS, and permeabilized with 0.1% (v/v) Triton X‐100 PBS for 10 min. After additional washes, unspecific binding sites were blocked with 2% normal goat serum and 1% BSA in PBS for 1 h at RT. Primary antibodies EEA1 (eBioscience, 14‐9114‐82; used at 1:1,000 dilution); LAMP2 (SouthernBiotech, 9840‐01; 1:50) were incubated 1 h in 3% BSA and washed as before. Secondary Alexa Fluor antibodies (Invitrogen) were incubated at a 1:1,000 dilution for 1 h at RT. Nuclei were counterstained with DAPI nuclear stain (Sigma‐Aldrich) together with the secondary antibody. Coverslips were mounted with Prolong Gold Anti‐Fade mounting medium (Invitrogen).
9
Visualizing APP Membrane Localization
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Membrane sheets were generated from cells expressing GFP-labeled APP or APP-TMS 21 h after transfection. Extracellular epitopes were stained by incubation of living cells for 2 h at 4 °C employing a mouse monoclonal antibody raised against β-amyloid amino acids 1 to 16 (diluted 1:100 in 1% BSA–PBS; clone 6E10; catalog no.: SIG-39320; Covance). After three washing steps with 0.5% BSA–PBS at 4 °C, cells were incubated with goat antimouse STAR RED (diluted 1:200 in 1% BSA–PBS; catalog no.: STRED-1001; Abberior Instruments) for 2 h at 4 °C. Then, membrane sheets were generated, fixed, quenched, and permeabilized as described previously. After blocking with 4% BSA–PBS, membrane sheets were stained for GFP using an Atto594-labeled GFP-Booster (catalog no.: gba594-100; ChromoTek) for 1 h at RT. Samples were washed three times in PBS, and cover slips were mounted on microscopy slides using ProLong Gold antifade mounting medium (catalog no.: P36930; Invitrogen), followed by imaging employing confocal microscopy.
10
Subcellular Localization Analysis
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Cells were seeded at a density of approx. 80% confluence on glass cover slips in a 6 cm diameter cell culture dish. After 12 hours, medium was replaced with fresh medium containing indicated inhibitor or vehicle. After 30 min, cells were transfected with the given plasmids. When needed, the medium was replaced after 4 hours with fresh medium containing 4-OHT or ethanol respectively, together with the inhibitors or vehicle as given. After 24 hours, cover slips were removed, washed in cold PBS, fixed 15 min. with 4% paraformaldehyde / PBS, washed, permeabilized for 10 min. with 0.1 % TritonX-100 and blocked for 1 hour in FBS. The remaining cells in the culture dish were processed for Western blot analysis. Cover slips were incubated with primary antibody diluted 1:100 in 50 % FBS / PBS / 0.05 % Tween-20 for one hour, washed and incubated with Alexa546 or Alexa488 labeled secondary antibody (dilution 1:500, Invitrogen, Freiburg, Germany) for 30 min. After washing, cover slips were mounted on a glass slide using Prolong Gold antifade mounting medium (Invitrogen). Images were acquired on a Leica SP5 confocal laser scanning microscope using a 630× oil immersion objective (Leica, Wetzlar, Germany).
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