Tumors tissue array (Alenabio, www.alenabio.com ) were fixed in 10 % formalin, embedded in paraffin, and followed by standard dewaxing procedures. Cells were cultured on the cover slips and fixed with freezing methanol after washing twice in PBS. The cover slips or tumors tissue array were then washed three times by PBS and blocked in PBS with 1 % BSA for 10 min or 1 h. The cover slips or tumor tissue arrays were hybridized with first and second antibodies for 1 h, respectively. Then, the slips were mounted with prolong anti-fade kit (Invitrogen) and observed with fluorescent microscopy. The arrays used for staining are as follows: H3K9me2 - BR243K, BR243L, BR243M, BR724, BR725, BCN963a; H3K9me3 - BR243B, BR243D, BR243K, BR243L, BR243A, BC081120, BCN963a; and KDM3A - BR724, BR725.
Prolong antifade kit
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany
The Prolong Antifade Kit is a reagent designed to maintain the fluorescence of fluorophore-labeled samples during microscopic observation. The kit helps preserve the signal-to-noise ratio and prevent photobleaching of the fluorescent dyes.
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Lab products found in correlation
Lsm 710 confocal microscope, by Zeiss (10 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (10 mentions)
Lsm 710, by Zeiss (5 mentions)
Lsm 510 meta confocal microscope, by Zeiss (4 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (4 mentions)
Lsm 880, by Zeiss (4 mentions)
Tcs sp5, by Leica (3 mentions)
Bovine serum albumin (bsa), by Merck Group (3 mentions)
Tcsnt sp2 confocal microscope, by Leica (3 mentions)
Normal horse serum, by Jackson ImmunoResearch (3 mentions)
Axiovision v4, by Zeiss (3 mentions)
Qimaging camera, by Nikon (2 mentions)
Alexa fluor 594 goat anti rabbit igg, by Thermo Fisher Scientific (2 mentions)
Hamamatsu orca er monochrome camera, by Hamamatsu Photonics (2 mentions)
In situ cell death detection kit, by Roche (2 mentions)
Ix81 inverted microscope, by Olympus (2 mentions)
Fv 1000 laser scanning confocal system, by Olympus (2 mentions)
Orca er monochrome camera, by Hamamatsu Photonics (2 mentions)
Paraformaldehyde (pfa), by Merck Group (2 mentions)
Paraformaldehyde (pfa), by Thermo Fisher Scientific (2 mentions)
118 protocols using prolong antifade kit
1
Immunofluorescence Analysis of Histone Modifications
2
Histological Analysis of Spinal Cord and Brain
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At 6 months after cell delivery, rats were deeply anesthetized with pentobarbital and transcardially perfused with heparinized saline (200 ml) followed by 4% paraformaldehyde in PBS (250 ml‐rats). The spinal cords and brains were then dissected and imaged in situ using Spectrum optical imaging system (Xenogen, Alameda, California). Sequences were acquired at excitation wavelength 465 nm and emission wavelength 520 nm. Medium binning was used, and the exposure time was 3 seconds. Images were analyzed using Living Image 4.3.1 (Xenogen) software. The signals were calculated using fixed volume region of interests (ROIs). After that, spinal cords and brains were cryoprotected in 30% sucrose PBS until transverse or longitudinal sections (30‐μm‐thick) were cut on a cryostat and stored in PBS. Sections were then stained with neuron‐specific or glia‐specific primary antibodies (Table 1 ) using the previously reported staining protocol.5 Then, staining sections were mounted on slides, dried at room temperature, and covered with a Prolong anti‐fade kit (Invitrogen). In vitro‐induced and 4% paraformaldehyde‐fixed hNSCs were stained using neuron or glia‐specific primary antibodies (Table 1 ).
Fluorescence images were captured using a Zeiss Imager M2 microscope and confocal images were taken using an Olympus FV1000 microscope.
Fluorescence images were captured using a Zeiss Imager M2 microscope and confocal images were taken using an Olympus FV1000 microscope.
3
Immunofluorescence Analysis of Cell Junctions
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Cells grown on Transwell pore filters were fixed with 4% paraformaldehyde in PBS for 20 min. Cells were incubated with rabbit anti-B16S antibody before permeabilization. After permeabilization with 0.5% Triton X-100 in PBS for 5 min, monolayers were incubated with primary antibodies, mouse anti-E-cadherin (BD Biosciences) or anti-ZO-1 (Zymed) monoclonal antibodies, probed with secondary antibodies coupled to Alexa488 (Invitrogen) or Cy3 (Jackson Immunoresearch), and mounted with ProLong Antifade kit (Invitrogen). All procedures were carried out at room temperature. Images were captured with an Olympus IX71 microscope equipped with a LCPlanFl 40×/0.60 objective (Olympus) and a CSU-X1 confocal scanner unit (Yokogawa). Images were analyzed with MetaMorph imaging software (version 7.7.10.0, Universal Imaging).
4
Measuring Intracellular Superoxide Anion
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MitoSOX™ Red (Invitrogen, Burlington, ON, Canada) was used to estimate intracellular superoxide anion production as already described [42 (link),43 (link),46 (link)]. Neuronal PC12 cells were seeded at 25,000 cells/cm2, differentiated and treated on collagen-coated circular glass coverslips. After treatment, NGF-differentiated PC12 cells were washed with Hank’s buffered salt solution (HBSS) and incubated for 10 min at 37 °C with a 5 µM solution of MitoSOX™ Red. Nuclei were counterstained with Hoescht 33342 (5 μg/mL) for 15 min at 37 °C, and then cells were fixed with 4% paraformaldehyde (PFA), mounted on glass slides with Prolong Antifade kit (Invitrogen), examined under a Leitz Orthoplan fluorescence microscope (Leica, Wetzlar, Germany) and photographed with a QImaging camera (Nikon, Mississauga, ON, Canada). Fluorescence intensity was measured using NIS Elements 2.2 software (Nikon, Mississauga, ON, Canada).
5
Placental Tissue Injury and Proliferation
6
Immunocytochemical Analysis of Glial Cells
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The cells were fixed in 4% paraformaldehyde (Sigma) in 1× PBS for 20 min, washed three times in PBS, and then blocked with 1× PBS containing 1% bovine serum albumin and 0.25% Triton X-100. The same solution was used during the incubations with the primary antibodies. The cells were incubated with antibodies overnight at 4°C. Fluorochrome-conjugated secondary antibodies were used for immunodetection. The primary antibodies were mouse antiglial fibrillary acidic protein (anti-GFAP) 1:1000 (Abcam) and rabbit anti-Galactocerebroside (anti-GalC) 1:500 (Abcam). The secondary antibodies were donkey anti-mouse conjugated with Alexa Fluor 568 1:1000 (Invitrogen); donkey anti-rabbit conjugated with Alexa Fluor 488 1:1000 (Dianova). Nuclear counterstaining was performed with 4′,6′-diamidino-2-phenylindole dihydrochloride hydrate at 0.25 μg/ml (Sigma). Coverslips were mounted onto glass slides using a Prolong Antifade kit (Invitrogen). The cells were quantified and photographed using an Olympus IX81 fluorescent microscope equipped with a Hamamatsu digital camera. For each culture condition, ten randomly selected fields were photographed, and the frequency of selected cellular markers was determined for every condition in three independent experiments.
7
Hemolymph Cell Immunofluorescence Assay
8
Immunocytochemical Staining Protocol
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Immunocytochemical studies were performed as described previously [25 (link)]. Cells were washed with PBS and fixed in 3.7 % formaldehyde (in PBS) for 20 min at 4 °C. Cells were permeabilized with PBS containing 0.2 % Triton X-100 for 5 min, blocked with 5 % BSA for 1 h and then washed three times with PBS. Incubation with primary antibody was carried out for 1 h at room temperature. Excess antibody was removed by washing three times with PBS. This was followed by incubation with an appropriate fluorophore-labeled secondary antibody for 1 h at room temperature in an area protected from light. After removing excess antibody by washing three times with PBS, mounting was performed using a ProLong Antifade Kit (Invitrogen). Images were obtained by fluorescence microscopy (Axio Imager M1; Zeiss, Oberkochen, Germany).
9
Fluorescent Cell Imaging on Coverslips
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Cells were plated on poly-L-lysine coated glass coverslips, and then fixed with 4% paraformaldehyde for 15min. Coverslips were mounted on glass slides using the Prolong Anti-fade kit (Invitrogen), and cells were imaged using a laser-scanning confocal Leica TCS SP5 microscope with 40× objective. Consecutive sections were sequentially scanned with 488 nm lasers, and sections were imaged at 0.97 μm intervals.
10
Immunodetection of LBH in Mouse Cochlea
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Cochleae were perfused with 4% formaldehyde in phosphate buffered saline (PBS) and treated with 0.2% Triton X-100/PBS. Goat serum (10%) was used to block nonspecific binding. The tissue was then incubated with an anti-LBH antibody (Sigma, Lot# HPA034669) and washed with PBS, followed by incubation with secondary antibodies (Life Technologies, Lot# 1579044). A sample was mounted on glass slides with antifade solution (Prolong Antifade Kit, Invitrogen, Carlsbad, CA) before imaging on a Leica Confocal Microscope (Leica TCS SP8 MP). Three cochleae from three adult (P28-P32) mice were used for immunodetection. Two adult cochleae were used as negative control (using only secondary antibody).
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