Immunofluorescence (IF) experiments were performed using standard procedures. Seven-micrometer lung sections were fixed in 4% paraformaldehyde, permeabilized with 0.2% Triton X-100, and treated with Super Block for blocking (Sytek Laboratories, West Logan, UT). Antibodies were diluted in buffered diluent (Immco Diagnostics, Buffalo, NY). Primary antibodies included either rabbit anti-HDAC1 1° (1:50, Abcam) or rabbit anti-phosphorylated-HDAC1 1° (1:200, Abcam) and donkey anti-rabbit Alexa-Fluor 594 2° antibody (1:500, Abcam). Slides were mounted with Prolong Gold with Dapi (Life Technologies, Carlsbad, CA). Pictures were taken at 20× to 40× using an Axiovert 200M fluorescence microscope (Zeiss, Oberkochen, Germany). n = 4 to 6 per group. Alveolar macrophages were isolated from bronchoalveolar (BAL) fluid and prepared for IF experiments, as described previously.26 (link) Standard IF procedures were followed, using 4% paraformaldehyde fixation, 0.2% Triton X-100 permeabilization, superblock blocking, anti-phosphorylated-HDAC1 (1:100, Abcam) 1° antibody, and donkey anti-rabbit AlexaFluor 594 (1:500, Abcam) 2° antibody. Slides were mounted with Prolong Gold with Dapi (Life Technologies). Pictures were taken at 100× using an Axiovert 200M fluorescence microscope (Zeiss) n = 5 to 7 per group.
Prolong gold with dapi
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany, Japan, United Kingdom
Prolong Gold with DAPI is a reagent used for mounting and preserving fluorescently labeled samples for microscopy. It contains the fluorescent dye DAPI, which binds to DNA and emits a blue fluorescence, allowing for the visualization of nuclei. The reagent helps to maintain the brightness and stability of the fluorescent signal over time.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 488, by Thermo Fisher Scientific (18 mentions)
Triton x 100, by Merck Group (13 mentions)
Paraformaldehyde (pfa), by Electron Microscopy Sciences (10 mentions)
Lsm 710, by Zeiss (10 mentions)
Lsm 900 confocal microscope, by Zeiss (9 mentions)
Superfrost plus slide, by Thermo Fisher Scientific (7 mentions)
Bovine serum albumin (bsa), by Merck Group (6 mentions)
Normal donkey serum (nds), by Jackson ImmunoResearch (6 mentions)
Alexa fluor 568, by Thermo Fisher Scientific (6 mentions)
Alexa fluor secondary antibody, by Thermo Fisher Scientific (6 mentions)
Lsm 780 confocal microscope, by Zeiss (6 mentions)
Axiocam mrc, by Zeiss (6 mentions)
Alexa fluor 594, by Thermo Fisher Scientific (5 mentions)
Alexa 488, by Thermo Fisher Scientific (5 mentions)
Saponin, by Merck Group (5 mentions)
Alexa fluor conjugated secondary antibody, by Thermo Fisher Scientific (5 mentions)
Dm5000 microscope, by Leica (5 mentions)
Sp5 confocal microscope, by Leica (4 mentions)
Paraformaldehyde (pfa), by Thermo Fisher Scientific (4 mentions)
Lsm 510 meta, by Zeiss (4 mentions)
391 protocols using prolong gold with dapi
1
Immunofluorescence Imaging of HDAC1 and Phospho-HDAC1
2
Immunofluorescent Staining of Mouse and Human Tissue
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All mouse tissue was from animals transcardially perfused with 4% paraformaldehyde, and was post-fixed for 2 h in 4% paraformaldehyde before being cyroprotected overnight in 30% sucrose. All human tissues were formalin-fixed for the duration between harvest and arrival in laboratory (16–24 h), after which the tissue was cyroprotected overnight in 30% sucrose. Both mouse and human tissue was cut as 25 μm thick sections on a cryostat. For immunofluorescent staining, sections were incubated overnight with primary antibodies diluted in phosphate buffered saline. Primary antibody manufacturers and dilutions are provided in Table 2 . Sections were then washed and incubated for 1 h with AlexaFluor (488, 594, or 647) secondary antibodies (Life Technologies) that were diluted at 1:400 in phosphate buffered saline. After removing the secondary antibodies, mouse sections were washed, dried and cover-slipped using Prolong Gold with DAPI (Life Technologies). Human sections were immersed in 0.02% Sudan Black in 70% ethanol for 20 min, then rinsed with 70% ethanol before being cover-slipped using Prolong Gold with DAPI (Life Technologies).
3
Immunofluorescence Analysis of HAS2 and VDR
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WT145 and KOhVDR cells were seeded into 4-well chamber slides at a density of 20,000 cells/well and treated the next day with 100 nM 1,25D3 or vehicle. 48 hours after treatment, slides were fixed and permeabilized with ice cold methanol for 10 minutes, rinsed with PBS, and blocked with Dako Protein Block for 1 hour at room temperature. Slides were stained with rabbit polyclonal HAS2 antibody (H-60, SantaCruz Biotechnology) overnight at 4°C. After washing with PBS, slides were incubated with AlexaFluor 488 secondary antibody (Life Technologies), washed and coverslipped with ProLong Gold with DAPI (Invitrogen). Hs578T cells were seeded in chamber slides, fixed in ice cold methanol and immunostained for HAS2 (4E7, Abcam), VDR (D6, Santa Cruz) or HA (bHABP). After primary antibody incubations, slides were incubated with appropriate secondary antibodies (Life Technologies) and coverslipped with ProLong Gold with DAPI (Invitrogen). Imaging was performed on a Leica DMI6000 microscope with a TCS SP5 confocal laser scanner using Leica Application Suite AF version 2.6.0.7266 software or on a Zeiss Axioskop 2 microscope equipped with Axiovision software.
4
Immunofluorescence Staining of Cells and Embryos
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Immunofluorescence staining was performed by fixing cells with 4% Paraformaldehyde/PBS for 10 min at 4 °C, then permeabilized with 0.1% Triton/PBS and blocked with 5% BSA/PBS before incubating with the primary antibodies. The list of antibodies used in this study and relative dilutions are provided in Supplementary Table 2 . Samples were rinsed with PBS, blocked with 5% BSA/PBS and then incubated with the secondary antibody. After washing, samples were mounted on the slides using Prolong Gold with DAPI (Invitrogen). Embryo cryosections (12-µm thick) were washed with PBS + 0.1%Tween (PBST), incubated for 1 h at RT in Permeabilization-Blocking solution (5% Normal Donkey Serum (Jackson Immunoresearch Laboratories), 0.1% Triton X-100 in 1× PBS) and then overnight at +4 °C with primary antibodies diluted in Antibody Diluent (Dako). Slides were washed three times with PBST, incubated with secondary antibodies diluted in Antibody Diluent (Dako) for 1 h at RT, washed again three times with PBST and briefly dried before mounting using Prolong Gold with DAPI (Invitrogen). Pictures were acquired with Axioimager M1 fluorescence microscope or LSM 510 Meta confocal microscope (Zeiss).
5
Visualizing Aβ Uptake in Macrophages
6
Immunofluorescence Staining of HEK293FT Cells
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HEK293FT cells were transfected as described. 24 hr post-transfection, cells were detached using trypsin treatment and 8 x 105 cells added to 6-well plates containing poly-L-lysine-coated glass coverslips. After 24 hr, wells were washed twice with PBS prior to cell fixation using 4% paraformaldehyde in fixative buffer (10 mM KCl, 274 mM NaCl, 8 mM NaHCO3, 0.8 mM KH2PO4, 4 mM MgCl2, 10 mM PIPES pH 7.2, 4 mM EGTA, 11 mM glucose) for 5 min at 37°C. Cells were permeabilised with 0.15% Triton-X 100 in fixative buffer for 20 min and washed once with 0.1 M glycine and four times with TBS for 10 min each. Coverslips were blocked with 1% BSA in TBS for 20 min. Coverslips were washed twice with 1% BSA in TBS for 10 min, twice with TBS for 5 min, and mounted onto glass slides using Prolong Gold with DAPI (Thermo Fisher). Cells were imaged on a Zeiss LS 780 confocal microscope.
7
Chromosome Analysis of Haploid and Diploid Plants
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Chromosome analysis was carried out as described in (44 (link)). Briefly, root tips were collected from the haploid and diploid plants, incubated in a chamber with nitrous oxide for 3 hours, and fixed with 90% acetic acid. Root tips were cut with a razor blade and digested in an enzyme solution (1% pectolyase Y-23 and 2% cellulase Onozuka R-10) at 37°C for 50 min. The root section was washed in ethanol and then immersed in 90% acetic acid. A metal pick was used to crush the roots tips, and 10 μl of the cell suspension was dropped onto microscope slides. Slides were dried and mounted with a glass coverslip using ProLong Gold with DAPI (Thermo Fisher Scientific, catalog no. P36931). Slides were imaged on a Zeiss Axio Imager M1 fluorescence microscope with a 63× Plan-Apochromat oil objective, and SlideBook software (Intelligent Imaging Innovations, Denver, CO, USA) was used to analyze the data.
8
Quantifying Muscle Stem Cell Activation
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Muscle cryosections were permeabilized with 0.5% triton X-100 for 10 min, washed with PBS, and underwent heat-mediated antigen retrieval with EDTA buffer (1 mM EDTA, pH 8.0; 0.05% Tween 20). Slides were washed with PBS and blocked for 1 h at room temperature with 3% BSA/PBS/0.1% Triton X-100. Sections were stained with Pax7 rabbit polyclonal antibody (1/100; Thermo #PA1–117) overnight at 4 °C, washed with PBS, and stained with Alexa Fluor-647-conjugated Goat anti-rabbit IgG (1/300; Thermo) for 1 hour at room temperature. Slides were washed with PBS and coverslips were mounted with prolong gold with DAPI (Thermo). Whole cryosections were imaged using an Axio Scan z1 slide scanner (Zeiss) and processed with Zen software. Two cryosections per mouse were used for quantification of the number of pax7 positive cells per 10μm section with Fiji.
9
Immunohistochemical Analysis of TERT
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Gastrocnemius cryosections were permeabilized with 0.5% triton X-100 for 10 min, washed with PBS, and blocked with 3% bovine serum albumin (BSA; GeminiBio) in PBS for 1 h at room temperature. Primary antibody to TERT (1/100; RayBiotech # 144–64552–50) was incubated in blocking buffer overnight at 4 °C. Slides were washed the following day with PBS and stained with Alexa Fluor-conjugated goat anti-rabbit 555 secondary antibody (1/300; Thermo #A21428) at room temperature for 1 hour. Slides were washed with PBS and coverslips were mounted with prolong gold with DAPI (Thermo). Slides were imaged on a Nikon-Ni widefield microscope using a Nikon DS-F12 color camera.
10
Immunofluorescence Analysis of YBX1 and YBAP1
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Cells were cultured on 12 mm round coverslips (corning) and were fixed with 4% EM-grade paraformaldehyde (Electron Microscopy Science, Hatfield, PA) in PBS pH7.4 for 10 min at room temperature. Cells were then washed 3 x with PBS for 10 min each, treated with permeabilizing buffer (10% FBS in PBS) containing 0.1% saponin for 20 min and treated in blocking buffer for 30 min. Subsequently, cells were incubated with primary antibodies in permeabilizing buffer for 1 hr at room temperature, washed 3 x with PBS for 10 min each and incubated with secondary antibodies in permeabilizing buffer for 1 hr at room temperature and finally washed 3 x with PBS for 10 min each. Cells were mounted on slides with Prolong Gold with DAPI (Thermo Fisher Scientific, P36931). Primary antibodies used in the immunofluorescence studies were as follows: anti-YBX1 (Abcam, ab12148), YBAP1 (Santa Cruz Biotechnology, sc-271200). Images were acquired with Zeiss LSM900 confocal microscope and analyzed with the Fiji software (http://fiji.sc/Fiji ).
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