Yale pathology kindly provided assistance with embedding, sectioning of lung tissue. A pulmonary pathologist reviewed the slides blinded and identified immune cell infiltration and other related pathologies. Paraffin embedded lung tissue (fixed in 4% paraformaldehyde for no more than 24 hours) sections were deparaffinized in xylene and rehydrated. After antigen retrieval with 10 mM Sodium Citrate pH 6 and permeabilization with 0.1% Triton-X for 10 min the slides were blocked with 5% BSA in PBS with 0.05% Tween 20 for an hour. Then the samples were stained with primary antibodies against SARS-CoV-2-dsRNA; SARS-CoV2-RNA-dependent RNA Polymerase, SARS-CoV-2-Spike, human CD68, human ACE2 their isotype controls diluted in 1%BSA overnight at 2–8 °C. The next day, the samples were washed and incubated with fluorescent secondary antibodies. After washes, samples were treated with TrueBlack lipofuscin autofluorescence quencher for 30 seconds and mounted on DAPI mounting media (Sigma). Images were acquired using Keyence BZ-X800 Fluorescence Microscope or Nikon ECLIPSE Ti Series Confocal Microscope. Pseudo-colors were assigned for visualization.
Dapi mounting media
Manufactured by Merck Group
Sourced in United States
DAPI mounting media is a laboratory product used to prepare microscope slides for fluorescence imaging. It serves as a mounting medium that preserves and protects the fluorescent signals of labeled samples, allowing for clear visualization and analysis under a microscope.
Automatically generated - may contain errors
Lab products found in correlation
Trueblack lipofuscin autofluorescence quencher, by Merck Group (2 mentions)
Bz x800 fluorescence microscope, by Nikon (2 mentions)
Eclipse ti series confocal microscope, by Nikon (2 mentions)
Axio observer z1 microscope, by Zeiss (2 mentions)
Ab150075, by Abcam (2 mentions)
Fv3000 confocal microscope, by Olympus (2 mentions)
Ab216484, by Abcam (1 mentions)
I 1000, by Vector Laboratories (1 mentions)
Nis element software, by Nikon (1 mentions)
Acetylated α tubulin, by Merck Group (1 mentions)
T6793, by Merck Group (1 mentions)
I 5000, by Vector Laboratories (1 mentions)
Af1589, by R&D Systems (1 mentions)
Sirt2 h 95, by Santa Cruz Biotechnology (1 mentions)
Alexa fluor, by Thermo Fisher Scientific (1 mentions)
Duolink kit, by Merck Group (1 mentions)
Axio observer 7 microscope, by Olympus (1 mentions)
Flouview3000 confocal microscope, by Olympus (1 mentions)
Mitotracker red cmxros, by Thermo Fisher Scientific (1 mentions)
Digoxigenin, by Merck Group (1 mentions)
14 protocols using dapi mounting media
1
Multimodal Lung Tissue Analysis
2
Osteocyte Immunostaining in Maxillary Molar
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Antigen retrieval was carried out in 10 mM of citric acid, pH 6.0, at 100 °C for one hour. Sections were incubated with primary antibody to IFT80 (PAB27850; Abnova, Taipei, Taiwan), acetylated α-tubulin (T6793; Sigma Aldrich, St. Louis, MO, USA), RANKL (ab216484; Abcam, Cambridge, MA, USA), and sclerostin (AF1589; R&D Systems, Minneapolis, MN, USA) overnight at 4 °C, as well as the matched negative control (I-1000 or I-5000; Vector Laboratories, Inc., Newark, CA, USA). HRP-conjugated secondary antibodies (705-035-147 or 111-035-144; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA), Alexa Fluor™ 647 tyramide reagent (B40958; ThermoFisher, Waltham, MA, USA), or Alexa Fluor™ 488 tyramide reagent (B40953) and DAPI mounting media (Sigma-Aldrich, St. Louis, MO, USA) were used. Four to six images per sample were taken at 40× objectives and assessed with NIS-Element software (Nikon) to examine the numbers of immunopositive osteocytes, divided by the area or total osteocytes on the compression side of the distobuccal root of maxillary 1st molar. Images were examined by a double-blinded examiner, and the results were confirmed with a second examiner. For all experiments, capture times were determined, so that the control IgG had no immunofluorescence.
3
Immunofluorescence Assay for SIRT1 and SIRT2
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Cells were seeded in eight-well chamber slides and treated for 72 h. Media was
removed and cells were rinsed with PBS. Cells were fixed with 4%
paraformaldehyde for 15 min at room temperature followed by rinsing with PBS
(three times, 5 min each). The cells were then permeabilized using ice-cold
methanol and rinsed again with PBS as previously described. The cells were
blocked with 5% BSA solution for 1 h, followed by rinsing with PBS again as
previously described. Subsequently, 200 µl of primary antibodies: SIRT1 (B-10)
(Santa Cruz, Cat#sc-74504, mouse monoclonal) and SIRT2 (H-95) (Santa Cruz,
Cat#sc-20966, rabbit polyclonal) were added to the cells and incubated at 4°C
overnight. Next, the cells were rinsed with PBS followed by incubation with
secondary antibodies (anti-mouse or anti-rabbit IgG, highly cross-adsorbed
secondary antibody, Alexa Fluor, Thermo Fisher, USA) for 1 h at room
temperature. Cells were washed in PBS three more times (5 min each). The slides
were removed from the chamber casket and mounted using Fluoroshield with DAPI
mounting media (Sigma). Cells were viewed and imaged using an inverted
fluorescent microscope (BX41, Olympus).
removed and cells were rinsed with PBS. Cells were fixed with 4%
paraformaldehyde for 15 min at room temperature followed by rinsing with PBS
(three times, 5 min each). The cells were then permeabilized using ice-cold
methanol and rinsed again with PBS as previously described. The cells were
blocked with 5% BSA solution for 1 h, followed by rinsing with PBS again as
previously described. Subsequently, 200 µl of primary antibodies: SIRT1 (B-10)
(Santa Cruz, Cat#sc-74504, mouse monoclonal) and SIRT2 (H-95) (Santa Cruz,
Cat#sc-20966, rabbit polyclonal) were added to the cells and incubated at 4°C
overnight. Next, the cells were rinsed with PBS followed by incubation with
secondary antibodies (anti-mouse or anti-rabbit IgG, highly cross-adsorbed
secondary antibody, Alexa Fluor, Thermo Fisher, USA) for 1 h at room
temperature. Cells were washed in PBS three more times (5 min each). The slides
were removed from the chamber casket and mounted using Fluoroshield with DAPI
mounting media (Sigma). Cells were viewed and imaged using an inverted
fluorescent microscope (BX41, Olympus).
4
Investigating Protein-Protein Interactions via PLA
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PLA was conducted to investigate direct protein-protein interactions within cells. Approximately 20,000 cells were seeded per well in 8-well chamber slides for this experiment. Following the designated treatments, cells were stained with 50 nM MitoTracker Red CMXRos (Thermo Fisher Scientific, USA) for 15 minutes under standard culturing conditions. After staining, cells underwent a washing step, and were then fixed with 4% PFA for 15 minutes at room temperature (RT). Subsequent steps included permeabilization in 0.2% Triton X-100 in 1X PBS for 10 minutes at RT, followed by PBS washing to remove any residual permeabilization agent. The in-situ PLA experiment was performed per the manufacturer’s guidelines, using the DuoLink kit (Sigma-Aldrich, USA). The primary antibodies used for the PLA are detailed in Supplementary Table 2. After the PLA procedure, coverslips were mounted using DAPI mounting media (Sigma-Aldrich, USA), and imaging was performed using either a Zeiss Axio Observer 7 microscope or an Olympus Flouview3000 confocal microscope.
5
Autophagy and Apoptosis Regulation
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Chloroquine, ammonium chloride, digoxin, digoxigenin, strophanthidin, DAPI mounting media, rapamycin, 3‐(4,5‐dimethylthiazol‐2yr)‐2‐5‐diphenyltetrazoliumbromide (MTT), actinomycin D, bafilomycin SMER28 and N‐acetylcysteine from Sigma (Poole, UK) were used.
Protein A magnetic beads were purchased from Thermo Scientific (East Riding of Yorkshire, UK). Antibodies against the following were used for immunoblotting: p62 (rabbit polyclonal; #P0067), LC3 (rabbit polyclonal; #L8918), Ubiquitin (rabbit polyclonal; #SAB1306222), NBR1 (rabbit polyclonal, #SAB2107031) from Sigma. p62 (mouse monoclonal; # sc‐28359), caspase‐3 (rabbit polyclonal; #sc‐136219) from Santa Cruz Biotechnology. Beta‐actin (mouse monoclonal; #ab8226), GFP (#ab1218), and LAMP1 (rabbit polyclonal; #ab24170) from Abcam. Cleaved caspase‐3 (rabbit polyclonal; #9661) from Cell Signalling (New England Biolabs). Keap1 (mouse monoclonal; #TA502059) and FLAG (mouse monoclonal; #TA50011‐100) from Origene).
Protein A magnetic beads were purchased from Thermo Scientific (East Riding of Yorkshire, UK). Antibodies against the following were used for immunoblotting: p62 (rabbit polyclonal; #P0067), LC3 (rabbit polyclonal; #L8918), Ubiquitin (rabbit polyclonal; #SAB1306222), NBR1 (rabbit polyclonal, #SAB2107031) from Sigma. p62 (mouse monoclonal; # sc‐28359), caspase‐3 (rabbit polyclonal; #sc‐136219) from Santa Cruz Biotechnology. Beta‐actin (mouse monoclonal; #ab8226), GFP (#ab1218), and LAMP1 (rabbit polyclonal; #ab24170) from Abcam. Cleaved caspase‐3 (rabbit polyclonal; #9661) from Cell Signalling (New England Biolabs). Keap1 (mouse monoclonal; #TA502059) and FLAG (mouse monoclonal; #TA50011‐100) from Origene).
6
Immunofluorescence Staining of Cell Markers
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USCs at passage three were cultured overnight in a 24–well plate over covered glass at 2.6 × 104 cells/cm2. Cells were then washed twice before fixing with 4% paraformaldehyde for 30 min at room temperature, followed by permeabilization with 0.25 Triton × 100 for 10 min. Cells were washed three times for 5 min each, and non–specific binding was blocked by buffer containing bovine serum albumin (BSA) (10 mg/mL) and glycine (22.52 mg/mL) for 30 min at room temperature. Cells were incubated with mAb to CD40L, BAFFR, and isotype control (Supplementary Table S1 ), diluted in PBS containing BSA (10 µg/mL), overnight in humidified chamber at 4 °C. Cells were washed as described above and incubated with Goat anti–mouse conjugated to either Alexa 488 or 647 diluted in PBS–BSA according to manufacture instructions for 1 h at room temperature. Cells were washed four times as described and mounted using DAPI mounting media (Sigma-Aldrich, St. Louis, MO, USA) for confocal microscope visualization.
7
Immunofluorescence Staining of Reprogrammed LNCaP Cells
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For LNCaP and STM-reprogrammed LNCaP, cells were plated at 25,000 cells per well in 4 chamber slides coated with PEI. Cell were incubated for 48 hours before fixing in 1% PFA. Slides were washed once with PBS before permeabilization with 0.5% Triton (30 minutes at room temperature). Cells were washed again before blocking in 3% milk for one hour, and overnight incubation with BRN3A and NESTIN antibodies. Cells were washed briefly before 1 hour incubation with secondary antibodies, and mounted in DAPI mounting media (Sigma). For differentiation to N/NC lineages, 25,000 LNCaP-SL cells were plated in PEI coated 4 chamber slides and allowed to attach for 24 hours before medium was replaced with osteoblast, neuron, glial, or oligodendrocyte re-differentiation medium. Media was refreshed every 3 days, and at day 8 cells were fixed and stained as described above. Immunofluorescence imaging was carried out on an Axio Observer Z1 Microscope (Carl Zeiss). See Supplementary Materials and Methods for details of antibodies employed.
8
EV Uptake by B Cells: Labeling and Quantification
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To study EV uptake by B cells, the EVs were labeled with ExoGlow™ Protein EV Labelling Kit (System Biosciences, Palo Alto, CA, USA). Then, the stained EVs were added to 2.5 × 105 B lymphocytes and cultured in complete media at 37 °C and 5% CO2 for 24 h. The cells were then collected and washed twice, centrifuged 400× g, and either stained for CD19 and CD69 for analysis by flow cytometry or fixed with 2% formaldehyde for observation with a confocal microscope. The fixed cells were then cytospinned (Cytospin 4, Thermofisher, Waltham, MA, USA) at 5000× g for 10 min. The cell pellet was mounted using DAPI mounting media (Sigma-Aldrich, St. Louis, MO, USA) and visualized in a confocal microscope (Olympus FV3000 Confocal Microscope, Tokyo, Japan) using the Z–stack technique to elaborate on the location of the vesicles.
9
Immunofluorescence Analysis of CD54 Expression
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BM‐MSCs and USCs at passage three were cultured over coverslips, washed twice before fixing with 4% paraformaldehyde (30 minutes). Cells were washed (X3) and blocked with bovine serum albumin (BSA) (10 mg/mL) and glycine (22.52 mg/mL) (30 minutes) at room temperature. Cells were incubated with mAb to CD54 and isotype control diluted in PBS containing BSA (10 μg/mL), overnight in humidified chamber at 4°C. Cells were washed and incubated with anti‐mouse Alexa flour 647 according to manufacture instructions for 1 hour at room temperature. Cells were washed and mounted using DAPI mounting media (Sigma‐Aldrich) for confocal microscope visualization (Olympus FV3000 Confocal Microscope, Japan).
10
Multimodal Lung Tissue Analysis
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