Cells were seeded onto coverslips and cultured for 2 hr prior to fixing and staining. Thereafter cells were fixed with 4% electron microscopy grade paraformaldehyde (Electron Microscopy Sciences) for 10 min at RT and then permeabilized for 3 minutes with 0.2% Triton-X 100 (Millipore Sigma). Over-extracted cells were treated with 0.4% Triton-X 100 for 10 min. Cells were then washed three times with DPBS and stained overnight at 4°C with primary antibodies diluted in PBS. Next, they were washed twice with PBS for 5 min, incubated with secondary antibodies (diluted 1:1000) for 2 hr at RT in PBS. Actin filaments were stained with Alexa Fluor 488, 568 or 647 phalloidin (Life Technologies) for 30 min at RT in DPBS. Cells were washed three times with DPBS before mounting with Prolong Diamond (Life Technologies). The following antibodies were used: Rabbit anti-Tom 20 (cat# 11802-1-AP, Proteintech) 1:300 dilution; rabbit-anti-parkin (cat # PA5-13399, Invitrogen) 1:50 dilution, mouse anti-DLP1 (cat# 611113 Clone 8/DLP1 (RUO), BD biosciences) 1:100 dilution, Preabsorbed secondary antibodies used were anti-mouse IgG 647, anti-rabbit IgG 568 at a 1:1000 dilution. Coverslips were mounted onto slides using Prolong Diamond (Thermo Fisher). All slides were cured at RT in the dark for 2 days prior to imaging.
Prolong diamond
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany, Japan, Australia
Prolong Diamond is a mounting medium designed for fluorescence microscopy. It is formulated to preserve fluorescent signals and protect samples from photobleaching.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (38 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (19 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (16 mentions)
Hoechst 33342, by Thermo Fisher Scientific (14 mentions)
Bovine serum albumin (bsa), by Merck Group (13 mentions)
Lsm 780 confocal microscope, by Zeiss (13 mentions)
Triton x 100, by Merck Group (12 mentions)
Lsm 710 confocal microscope, by Zeiss (10 mentions)
Paraformaldehyde (pfa), by Thermo Fisher Scientific (6 mentions)
Paraformaldehyde (pfa), by Merck Group (6 mentions)
Alexa fluor conjugated secondary antibody, by Thermo Fisher Scientific (6 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (6 mentions)
Lsm 700, by Zeiss (6 mentions)
710 confocal microscope, by Zeiss (6 mentions)
Zen black, by Zeiss (5 mentions)
Nis elements software, by Nikon (5 mentions)
Prolong gold, by Thermo Fisher Scientific (5 mentions)
Alexa fluor 647, by Thermo Fisher Scientific (5 mentions)
Vectashield, by Vector Laboratories (5 mentions)
Paraformaldehyde (pfa), by Electron Microscopy Sciences (4 mentions)
287 protocols using prolong diamond
1
Immunofluorescence Staining of Mitochondrial Proteins
2
Immunofluorescence Staining Protocol
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Cells were rinsed twice with phosphate-buffered saline (PBS) and fixed using 4% (vol/vol) paraformaldehyde/PBS for 15 min at room temperature. The fixative was removed, and the cells were permeabilized with 0.1% (vol/vol) Triton X-100 for 10 min at room temperature. The cells were rinsed twice with PBS and quenched with 0.2 M glycine for 10 min at room temperature. The cells were then rinsed with PBS, and coverslips were incubated in primary antibodies diluted in 25 μl of 1% bovine serum albumin (BSA)/PBS for 1 h at room temperature. After incubation with primary antibodies, the cells were washed three times with 0.1% BSA/PBS. Coverslips were incubated in secondary antibodies diluted in 25 μl of 1% BSA/PBS for 45 min at room temperature. The cells were washed twice with PBS and incubated for 5 min with DAPI (4′,6′-diamidino-2-phenylindole at 0.33 μg/ml in PBS. The coverslips were rinsed twice with PBS and Milli-Q water and mounted on cover slides with ProLong Diamond (Life Technologies). The cells were analyzed using a Zeiss LSM710 confocal microscope.
3
Immunofluorescence Staining of Brain Sections
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Mice were anesthetized by CO2 inhalation before perfusion with PBS containing 4% paraformaldehyde and 4% sucrose. Brains were harvested and post-fixed overnight in the same fixative and then stored at 4 °C in PBS containing 30% sucrose. Sixty μm-thick coronal sections were cut on a cryostat and processed for free-floating immunofluorescence staining. Brain sections were incubated with the indicated primary antibodies for 48 h at 4 °C followed by secondary antibodies (Invitrogen) for 24 h at 4 °C. The antibodies were diluted in 1X Tris Buffer Saline solution containing 10% donkey serum, 3% BSA, and 0.25% Triton-X100. Sections were then mounted on slides with Prolong Diamond (Life Technologies) before confocal microscopy. The list of antibodies is provided in Supplementary data 9 .
4
Quantifying MeCP2 Protein Recruitment
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NIH-3T3 cells were seeded on coverslips in 6-well plates (25,000 cells per well) and transfected with 2 μg plasmid DNA (pEGFPN1-MeCP2 alone or pEGFPN1-MeCP2 and pmCherry-TBL1X5 (link)) using JetPEI (PolyPlus Transfection). After 48 hours, cells were fixed with 4% (w/v) paraformaldehyde, stained with DAPI (Sigma) and then mounted using ProLong Diamond (Life Technologies). Fixed cells were photographed on a confocal microscope (Leica SP5) using LAS AF software (Leica). The number of co-transfected cells with TBL1X-mCherry recruitment to heterochromatic foci was determined for each MeCP2 construct. In total, 113-125 cells per construct were counted (from three independent transfection experiments). This analysis was performed blind. The total proportion of cells with TBL1X-mCherry recruitment by each mutant MeCP2 protein was compared to WT using Fisher’s exact tests.
5
Immunofluorescence Staining of Endothelial Cells
6
Cardiac Progenitor Cell Hypoxia Assay
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Passage 7 cardiac PCs were grown in a 96‐well plate (PerkinElmer Cell Carrier, Perkin Elmer, Waltham, MA, USA) until 90% confluent. Cells were washed with warm 1× DPBS and fixed with 4% paraformaldehyde (Sigma) for 30 min at room temperature. Cells were washed three times with 1× DPBS and permeablized with 0.1% Triton X‐100 (Sigma) for 10 min at room temperature. Cells were then blocked with SuperBlock (Thermo Fisher Scientific, Waltham, MA, USA) for 1 h at room temperature. After blocking, anti‐hypoxia‐inducible factor 1 alpha (HIF‐1α) antibody (Abcam, Cambridge, UK) diluted at 1 : 100 with SuperBlock was added and incubated at 4 °C overnight. Next day, cells were washed three times with wash buffer (0.1× SuperBlock + 1× DPBS). Secondary anti‐rabbit HRP (Cell Signaling Technologies, Danvers, MA, USA) diluted 1 : 1000 in SuperBlock was added and incubated for 2 h at room temperature in the dark. Secondary antibody was washed off three times with wash buffer. A 1 : 1000 dilution of 300 µm DAPI (Life Technologies, Carlsbad, CA, USA) stain was added on for 5 min at room temperature. Cells were washed three times with 1xDPBS and mounted with ProLong Diamond (Life Technologies) mounting media. Cells were imaged on an Opera Phenix confocal (PerkinElmer) at 40×.
7
Immunohistochemical Analysis of RA Synovium
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RA synovial tissues were obtained by biopsies from RA patients in the BEACON Birmingham early arthritis cohort, which is an early arthritis cohort recruiting patients with new onset arthritis prior to treatment with disease-modifying antirheumatic drugs. Synovial tissues for staining were frozen in OCT compound. Sections were made in 6 μm thickness, fixed with acetone, and frozen prior to use. Slides were rehydrated in phosphate-bufferred saline (PBS), blocked with 10% normal goat or horse serum in PBS for 10 min, and then incubated with primary antibodies, followed by secondary antibodies. Slides were mounted using ProLong Diamond (Life Technologies), and imaged using a Zeiss LSM 780 or 800 confocal microscope. Images were processed using Zen Black and Zen lite (Zeiss). Representative images were shown. Synovial tissues for histological analysis and hematoxylin and eosin staining were fixed in formaldehyde, then mounted in paraffin, sectioned, and stained by the Hospital Pathology service.
8
Immunofluorescence Analysis of DNA Damage
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Cells grown on glass coverslips (VWR) were fixed with 3.7% formaldehyde in PBS for 15 min and permeabilized with 0.5% Triton X-100 in PBS for 20 min. Samples were blocked in 3% BSA (Sigma Aldrich) in PBS before overnight incubation at 4 °C with primary antibodies: 1:400 S9.6 (gift from P. Huertas, CABIMER, Spain47 (link) or 1:200 anti-γH2AX (Abcam), followed by 1 h incubation at room temperature with the respective secondary antibodies conjugated to Alexa-Fluor 594, 647 or 488 (Thermo Fisher Scientific) and then 5 min staining at room temperature with 2 ng/μl of DAPI (Merck) in PBS. Coverslips were mounted in Prolong Diamond (Life Technologies) and visual acquisition was performed in a LSM510 AxioImager M1 microscope (Zeiss) using either a ×63 or a ×100 oil objective. Nuclear segmentation was based on DAPI staining. Statistical analyses were performed in Prism v5.0.4 (GraphPad Software) using the non-parametric Mann–Whitney rank sum test . *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
9
Immunofluorescence Staining Protocol
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Immunofluorescence was performed as previously described (Hicks et al., 2015 (link)). In brief, cells were fixed in 4% formaldehyde (Pierce, ThermoFisher) for 5 min at room temperature. The cells were permeabilized by washing in PBST (PBS +0.1% Triton X-100). The primary antibody was incubated in blocking buffer (PBS +5% Normal goat serum) overnight at 4°C. Next, the samples were washed three times in PBST and incubated for 1 h at room temperature with the fluorescent secondary antibody (Goat anti-mouse or Goat anti-rabbit antibodies conjugated with either Alexa488 or Alexa555, 1:400 dilution, Life technologies) in the same blocking buffer. The samples were then washed four times with PBST. In order to visualize nuclei, the cells were stained with DAPI. For visualizing the actin cytoskeleton Alexa633 conjugated Phalloidin was used (Life technologies; 1:400). For detecting biotinylated proteins, the samples were incubated with Streptavidin Alexa647 (Life technologies; 1:1,200). The Strep674 was added to the sample at the same time as the secondary antibody. The cells were mounted onto slides using Prolong Diamond (Life technologies).
10
SNAP-LGR5 and SNAP-FZD5 Colocalization
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HEK293T cells were grown on laminin‐coated glass coverslips. Cells were co‐transfected with 100 ng SNAP‐LGR5 or SNAP‐FZD5 and 150 ng myc‐NEDD4, HA‐NEDD4‐CS, myc‐NEDD4L, HA‐NEDD4L‐CA or pcDNA4 as a control with Fugene according to the manufacturer's instructions. After 24 h of transfection, cells were labelled with 1 μM SNAP‐Surface Alexa 488 (NEB) for 15 min at 4°C to block endocytosis. Cells were then immediately washed with fresh RPMI media and fixed in 4% paraformaldehyde. Cells were incubated with primary antibodies NEDD4 (Santa Cruz) or NEDD4L (Cell Signaling) for 1 h at RT followed by a secondary antibody conjugated to Alexa‐568 (Invitrogen) and DAPI (Sigma) in 2% BSA‐PBS (Roche). Cells were mounted in Prolong Diamond (Life technologies) and imaged using a DeltaVision Core system.
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