The immunofluorescence staining of cell surface or intracellular proteins was performed as above. Thereafter, cells were resuspended in 20 μL PBS after twice washing steps with PBS and covered on a slide with a thin layer. After air drying, cell layers were added with mounting solution (Thermo Fisher Scientific, USA) and covered by coverslips for fluorescence microscopy. Images were made at a magnification of ×600.
Mounting solution
Manufactured by Thermo Fisher Scientific
Sourced in United States, Japan
Mounting solution is a laboratory product designed to prepare and preserve samples for microscopic analysis. It is a clear, viscous liquid that is used to mount and secure specimens on microscope slides, allowing for their observation and analysis under a microscope.
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Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (4 mentions)
Eosin y solution, by Merck Group (2 mentions)
Lab tek chambered coverglass, by Thermo Fisher Scientific (2 mentions)
Evos fl, by Thermo Fisher Scientific (1 mentions)
Adi 950 233 0001, by Enzo Life Sciences (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Hematoxylin solution, by Thermo Fisher Scientific (1 mentions)
Igepal, by Merck Group (1 mentions)
A1r confocal system, by Nikon (1 mentions)
Anti brdu, by Merck Group (1 mentions)
Bromodeoxyuridine (brdu), by Merck Group (1 mentions)
Alexa 594 conjugated goat anti mouse igg, by Abcam (1 mentions)
Anti pax7, by Abcam (1 mentions)
Alexa 488 conjugated goat anti rabbit igg, by Abcam (1 mentions)
Rm2235, by Leica (1 mentions)
Dmi8 fluorescence microscope, by Leica (1 mentions)
Slide warmer, by Thermo Fisher Scientific (1 mentions)
Paraffin embedding center, by Leica (1 mentions)
Paraplast, by Leica (1 mentions)
Harris hematoxylin solution, by Merck Group (1 mentions)
21 protocols using mounting solution
1
Immunofluorescence Staining for Microscopy
2
Rhodamine B Skin Permeability Assay
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Rhodamine B, a hydrophilic dye, was applied to evaluate the permeability of the skin as described previously [41 (link)]. Treated tissues were incubated for 24 h and rinsed with PBS. 0.02% rhodamine B was applied for 2 h and gently washed with an autoclaved cotton swab. Then, cultured tissues were initially cut in 100-mm sections and immediately embedded in OCT compound (Sakura, Tokyo, Japan) using dry ice. Embedded samples were cut into 5-μm sections and incubated at room temperature for 40–60 min. Samples were washed in PBS twice for 3 min. After washing, 1:1,000 DAPI (Sigma, St. Louis, OU, USA) in PBS was applied for 5 min on slides which were pre-marked by a pen (Enzo, ADI-950-233-0001, Farmingdale, IL, USA), followed by three washes in PBS. Then, samples were covered by mounting solution (Thermo Scientific, Waltham, MA, USA), and immunofluorescence images were captured by EVOS-FL (Thermo Scientific, Fremont, CA, USA).
3
Hematoxylin-Eosin Staining of Retinal Sections
4
Immunocytochemistry of THP1 Cells
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THP1 cells were differentiated as mentioned above and placed on coverslips (Fisher Scientific). The cells were fixed using 4% PFA, followed by blocking and permeabilization with 0.1% Igepal (Sigma–Aldrich, Inc.) in DPBS with 2% BSA (Amresco, OH, United States). Primary antibodies diluted in DPBS with 2% BSA (Sangon Biotech, Shanghai, China) were applied overnight at 4 °C. The cells were subsequently washed four times with DPBS before being incubated with the appropriate secondary antibodies (Invitrogen) diluted in 2% BSA in DPBS. The coverslips were washed four times with DPBS before being mounted on slides using mounting solution (Thermo Fisher Scientific). Confocal images were acquired with a Nikon A1R confocal system.
5
Muscle Stem Cell Proliferation Assay
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Animals were given water with BrdU (0.8 mg/ml) (Sigma, Saint Louis, MO) for 3 days after injection and then animals were sacrificed and soleus were removed 14 d after immobilization. Muscles were fixed and incubated with 2 N HCl for 20 min. Samples were washed and sections were incubated with anti-BrdU (1:200; Sigma, Saint Louis, MO) and anti-Pax7 (1:200, Abcam) at 4°C overnight, followed by Alexa 594-conjugated goat anti-mouse IgG (1:500, Abcam) and Alexa 488-conjugated goat anti-rabbit IgG (1:500, Abcam). Finally, the sections were incubated with DAPI (Life Technologies, Japan) for 1 min, washed in PBS, and mounted in mounting solution (Thermo Fisher Scientific). Positive cells were counted under 20 high-power fields (HPFs) of 25 sections and the relative number of Pax7/BrdU-positive cells in HPF was noted.
6
Alcian Blue Staining of Paraffin Sections
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The pelleted cells were embedded in paraffin. After sectioning, slides were deparaffinized with xylene and hydrated with distilled water. After incubation of slides in 3% acetic acid for 3 minutes, the slides were stained with a 1% Alcian blue solution (in 3% acetic acid, pH 2.5) for 30–45 minutes. The slides were then washed with water for 2 minutes, dehydrated with xylene, and mounted with mounting solution (Thermo Fisher Scientific, Waltham, MA, USA).
7
Histopathological Analysis Protocol: Paraffin Sections
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For histopathological analyses, paraffin-embedded blocks were cut into 5 µm sections using a microtome (RM2235, Leica). Sections were deparaffinized with xylene (UN1307, Baker) three times, each for 20 min. Sections were dried at room temperature after two washes in 100% EtOH for 5 min each wash. For hematoxylin and eosin (H&E) staining, sections were stained with 0.1% Mayer's hematoxylin for 10 min and 0.5% eosin in 95% EtOH for 10 min. After H&E staining, sections were washed in distilled H2O until the eosin stopped streaking. They were then dipped in 50% EtOH 10 times, 70% EtOH 10 times, 95% EtOH for 30 s, and 100% EtOH for 1 min. Samples were then covered with mounting solution (Thermo Scientific) and diagnosed by a pathologist under a light microscope (BX43, Olympus).
8
Quantifying EV Internalization Kinetics
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To monitor the kinetics of EV internalization in vitro, EVs were labeled as per the protocol mentioned above. M1 cells (5 × 104) were grown on the round glass coverslip on 12-well plates and supplemented with 500 µL DMEM media mixed with 5 × 106 labeled EVs, and incubated for a range of time points between 0 and 24 h. After completion of time points, cells were washed with cold PBS and fixed with 4% paraformaldehyde, followed by permeabilization with 0.1% Triton X-100. Cells were stained with 4′,6-diamidino-2-phenylindole (DAPI) and mounted with mounting solution (ThermoFisher Scientific). Images were captured using a Leica DMi8 fluorescence microscope. The fluorescence intensity was estimated using ImageJ software. At least 100 cells for each time point were analyzed at a magnification of 40× to determine the fluorescence measure of EV uptake (Supplemental Figure S2 ).
9
Histological Processing of Brain Tissues
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Brain tissues were fixed in 4% neutral buffered paraformaldehyde, washed with tap water for overnight, dehydrated with graded series of ethanol from 70 to 100%, and cleaned with xylene. They were infilterated with paraplast (Leica, Wetzlar, Germany) and embedded using paraffin embedding center (Leica). Paraffin blocks were cut into 4 µm thicknesses using rotary microtome (Leica) and paraffin ribbons were mounted on slide glass. Sections were dried on slide warmer (Thermo Fisher Scientific, Waltham, MA, USA). They were dipped in xylene to remove paraffin, hydrated with graded series of ethanol from 100 to 70%, and washed with tap water. Sections were stained with Harris’ hematoxylin solution (Sigma-Aldrich) for 5 min and washed with tap water. They were subsequently dipped in 1% hydrochloric acid with ethanol, washed with water, and dipped in 1% ammonia water. Sections were washed with water, stained with eosin Y solution (Sigma-Aldrich) for 2 min, and washed with water. They were dehydrated with graded series of ethanol from 70 to 100%, cleaned with xylene, and coverslipped with mounting solution (Thermo Fisher Scientific). The stained tissues were observed and photographed under an Olympus microscope (Olympus, Tokyo, Japan).
10
Histological Analysis of 3D-RHPE Model
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For histological analysis of the 3D-RHPE model, the tissue was stained with hematoxylin and eosin (H&E). The tissue was sectioned (7-μm thickness) and de-paraffinized by washing with xylene three times for 1 min and 100%, 95%, 90%, 80%, and 70% ethyl alcohol for 30 s each. The sections were stained with Mayer's Hematoxylin (Merck, Darmstadt, Germany) for 60 sec and washed with tap water for 5 min. Sections were counterstained by rinsing with Eosin Y-solution (Merck) for 4 min, while dehydration was performed by sequentially washing with 95% ethyl alcohol, 100% ethyl alcohol, and xylene for 30 s each. Mounting solution (Thermo Fisher Scientific) was added to the slides, which were covered prior to examination by light microscopy using an Olympus CKX 41 microscope (Japan). To quantify melanin in the 3D-RHPE model, Fontana-Masson staining was performed on the formalin-fixed, paraffin-embedded tissue. The tissue section was treated with 2.5% aqueous silver nitrate solution for 10 min, 0.2% aqueous gold chloride for 1 min, and 5% aqueous sodium thiosulfate for 5 min. The number of pixels representing Fontana-Masson staining were normalized to the total amount of epidermal counterstain. The values were expressed as the mean value ± SD. Statistical analysis was performed using Student's t-test and the significance level was set to p < 0.05.
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