Double labeling with xylenol orange and calcein was used to study bone turnover during M. avium infection (14 (link)). Thus, 6 weeks post-infection, a saline solution of xylenol orange (Sigma-Aldrich, USA) was subcutaneously administered at a dose of 80 mg/kg. Two weeks later, a saline solution of calcein green (Sigma-Aldrich, USA) at a dose of 15 mg/kg was administered by the same route as xylenol orange. Three days later (at 8 weeks post-infection), mice were euthanized, and femurs and tibias were collected, fixed in 4% (v/v) paraformaldehyde (BioOptica, Italy) at 4°C, overnight. Then, the bones were washed 3 times with PBS1x for 10 minutes, followed by dehydration with PBS1x/30% (w/v) sucrose, at 4°C, overnight. Long bones were embedded in OCT (ThermoFisher, UK), snap-frozen in an ethanol/dry ice bath, and 20-µm sections were cut using the Kawamoto method (15 (link)), using a Leica CM 3050S cryostat (Leica Biosystems, Portugal). Sections were kept at -20°C, inside a silica gel chamber, for at least 12 hours. Then, slices were warmed to room temperature inside a silica gel chamber and mounted in a 30% (v/v) glycerin solution. Images were acquired on a Leica SP8 confocal microscope (Leica Microsystems, Germany), using LASX software (Leica Microsystems, Germany). Images were analyzed using the measurement tool of Imaris Software (Oxford Instruments, UK).
Paraformaldehyde (pfa)
Manufactured by Bio-Optica
Sourced in Italy
Paraformaldehyde is a solid polymer of formaldehyde, commonly used as a fixative in histological and cytological applications. It is a white, crystalline solid that releases formaldehyde gas upon heating or dissolution in water. Paraformaldehyde is an effective cross-linking agent, which helps preserve the structural integrity of cells and tissues during sample preparation for microscopy and other analytical techniques.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Merck Group (2 mentions)
Penicillin, by Merck Group (2 mentions)
Streptomycin, by Merck Group (2 mentions)
Xylenol orange, by Merck Group (1 mentions)
Leica cm3050s cryostat, by Leica Biosystems (1 mentions)
Las x software, by Leica Microsystems (1 mentions)
Leica sp8 confocal microscope, by Leica Microsystems (1 mentions)
Imaris software, by Oxford Instruments (1 mentions)
Fluoview microscope bx61, by Olympus (1 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (1 mentions)
Bleomycin, by Abcam (1 mentions)
Doxorubicin, by Merck Group (1 mentions)
Tissue tek oct, by Thermo Fisher Scientific (1 mentions)
Fluoroshield with dapi, by Vector Laboratories (1 mentions)
Gelred nucleic acid, by Biotium (1 mentions)
Prestoblue cell viability reagent, by Thermo Fisher Scientific (1 mentions)
Masson s trichrome, by Bio-Optica (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Hoechst 33342, by Merck Group (1 mentions)
6 protocols using paraformaldehyde (pfa)
1
Bone Turnover Analysis in Mycobacterium avium Infection
2
Cellular Uptake of CNC-Based Formulations
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Human mammary adenocarcinoma cells, MDA-MB-231, were used to perform the uptake assay.
Cells were cultured in DMEM (Sigma-Aldrich) complemented with 10% FBS (Sigma-Aldrich), 2 mM L-glutamine, and 100 U penicillin/0.1 mg/ml streptomycin, and maintained at 37°C in 5% CO 2 . Cells were seeded at a density of 15000 cells/cm 2 in 24-well plates, incubated with the different compounds, and the uptake efficiency was observed at 6, 24 and 48 h. CNC-SO 3 -and CNC-ALN were incubated at 70 µg/ml of CNCs. In additional experiments, CNC-DOX and free DOX were used at 0.5 µM of DOX. After incubation, cells were washed and fixed with 4% paraformaldehyde (Bio-Optica) for 45 min. The nuclei were stained with Hoechst-33258 (2 μg/ml in PBS) for 10 min. Cells were analyzed with Olympus Fluoview microscope BX61 with confocal
8 system FV500 (λ exc 405 nm for Hoechst-33258; λ exc 532 nm to visualize DOX; λ exc 635 nm to visualize Alexa Fluor 633-labelled CNCs). Image quantification was made through Image J software and for each experimental group 10 acquisitions were captured with 20x objective. The values express the ratio between the signal associated with CNCs (area) and the cell number for each field of view.
Cells were cultured in DMEM (Sigma-Aldrich) complemented with 10% FBS (Sigma-Aldrich), 2 mM L-glutamine, and 100 U penicillin/0.1 mg/ml streptomycin, and maintained at 37°C in 5% CO 2 . Cells were seeded at a density of 15000 cells/cm 2 in 24-well plates, incubated with the different compounds, and the uptake efficiency was observed at 6, 24 and 48 h. CNC-SO 3 -and CNC-ALN were incubated at 70 µg/ml of CNCs. In additional experiments, CNC-DOX and free DOX were used at 0.5 µM of DOX. After incubation, cells were washed and fixed with 4% paraformaldehyde (Bio-Optica) for 45 min. The nuclei were stained with Hoechst-33258 (2 μg/ml in PBS) for 10 min. Cells were analyzed with Olympus Fluoview microscope BX61 with confocal
8 system FV500 (λ exc 405 nm for Hoechst-33258; λ exc 532 nm to visualize DOX; λ exc 635 nm to visualize Alexa Fluor 633-labelled CNCs). Image quantification was made through Image J software and for each experimental group 10 acquisitions were captured with 20x objective. The values express the ratio between the signal associated with CNCs (area) and the cell number for each field of view.
3
Multimodal Analysis of HCC1954 Cell Line
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PCL (MW 60 kDa), sodium cacodylate, sucrose, acetonitrile, propidium iodide (PI), Hoechst 33342 (HOE), hexafluoroisopropanol (HFIP), doxorubicin, and bovine serum albumin (BSA) were obtained from Sigma Aldrich (Steinheim, Germany), and ethanol from VWR Chemicals Prolab (Fontenay-sous-Bois, France). Bleomycin was from Abcam (Cambridge, UK).
The HCC1954 cell line was provided by ATTC (Guernsey, UK). Fetal bovine serum (FBS), culture media, and supplements were obtained from Corning (Mediatech Inc., Manassas, VA, USA). The PrestoBlue™ Cell Viability Reagent and secondary antibody goat anti-mouse Alexa 488 were purchased by Thermo Fisher Scientific (Eugene, OR, USA), whereas paraformaldehyde, Masson’s trichrome, and Alcian blue staining kits by BioOptica (Milan, Italy). The TriReagent and the qPCRBIO SyGreen 1-Step Go Lo-ROX were purchased from PCRBIOSYSTEMS (London, UK), and Gel Red Nucleic Acid staining from Biotium (Hayward, CA, USA). Anti-CD44 antibody was provided by AbD Serotech (Kidlington, UK), and Fluoroshield with DAPI was from Vector Laboratories (Peterborough, UK). Tissue-Tek® OCT was purchased from Thermo Fisher Scientific (Waltham, MA, USA).
The HCC1954 cell line was provided by ATTC (Guernsey, UK). Fetal bovine serum (FBS), culture media, and supplements were obtained from Corning (Mediatech Inc., Manassas, VA, USA). The PrestoBlue™ Cell Viability Reagent and secondary antibody goat anti-mouse Alexa 488 were purchased by Thermo Fisher Scientific (Eugene, OR, USA), whereas paraformaldehyde, Masson’s trichrome, and Alcian blue staining kits by BioOptica (Milan, Italy). The TriReagent and the qPCRBIO SyGreen 1-Step Go Lo-ROX were purchased from PCRBIOSYSTEMS (London, UK), and Gel Red Nucleic Acid staining from Biotium (Hayward, CA, USA). Anti-CD44 antibody was provided by AbD Serotech (Kidlington, UK), and Fluoroshield with DAPI was from Vector Laboratories (Peterborough, UK). Tissue-Tek® OCT was purchased from Thermo Fisher Scientific (Waltham, MA, USA).
4
Confocal Analysis of hGMSCs Signaling
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To perform the Confocal Laser Scanning Microscope analysis, 6.4 × 103/well of hGMSCs were placed in 8-well culture glass slides and treated with different stimuli for 72 h (as mentioned above). Then, the cells were fixed with 4% of paraformaldehyde (PFA) (BioOptica, Milan, Italy) in PBS (0.1 M) (Lonza, Basel, Switzerland) for 1 h at room temperature, washed 3 times with PBS, permeabilized with 0.1% Triton X-100 (BioOptica) in PBS and blocked for 1 h at room temperature with 5% of skimmed milk in PBS. The primary antibodies anti-TLR4 (sc-293072, Santa Cruz Biotechnology, Dallas, TX, USA), anti-MyD88 (sc-74532, Santa Cruz Biotechnology), anti-NFκB p65 (sc-8008, Santa Cruz Biotechnology) and anti-NALP3 (sc-134306, Santa Cruz, Biotechnology) were diluted 1:200 and incubated overnight at 4 °C. Then, the secondary antibody Alexa Fluor 568 red fluorescence-conjugated goat anti-mouse (1:200) (A11031, Invitrogen, Eugene, OR, USA) was added to the samples for 1 h at room temperature, followed by the final incubation with 1:200 of Alexa Fluor 488 phalloidin green fluorescent conjugate (A12379, Invitrogen) and TOPRO (T3605, Invitrogen) mixed together. The images were obtained with a Zeiss LSM800 confocal system (Carl Zeiss, Jena, Germany) [49 (link)].
5
Evaluating αSyn Aggregation in Neuroblastoma
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For I-threo and IV-threo treatment, SK-N-SH neuroblastoma cell line stably overexpressing αSyn were used as an in vitro model of synucleinopathy as they exhibit αSyn aggregation, as it has been previously reported that three days αSyn overexpression is sufficient to induce protein aggregation [40 (link)]. Briefly, cells were seeded onto poly-D-lysine-coated glass cover slides in 24-well plates (10,000 cells/well) and maintained at 37 °C under a humidified atmosphere of 5% CO2 and 95% O2 in Dulbecco’s modified Eagle medium with 1000 mg glucose/L (Sigma-Aldrich/ Merck KGaA, Darmstadt, Germany), 10% heat-inactivated fetal bovine serum, 100 μg/mL penicillin, 100 μg/mL streptomycin and 0.01 μM non-essential amino acids (Sigma-Aldrich/ Merck KGaA, Darmstadt, Germany). When the cells reached 80% confluence, these were treated with I-threo 0.05, 0.1, 10 μM or with IV-threo 10 μM for 24 h, to assess the effect of the compounds on αSyn aggregation. Cells were then fixed with 4% PFA (Bio-Optica, Milan, Italy) for 15 min, at 4 °C (n = 3 replicates).
6
Primary Ventral Mesencephalic Cell Isolation and Culture
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Primary ventral mesencephalic cells from C57 BL/6 J 13.5 days embryos were isolated and cultured as previously described [3 (link)]. Briefly, after 10 min enzymatic dissociation, the single cell suspension was diluted in Neurobasal medium (Life Technologies, Carlsbad, CA, USA) added with 100 μg/mL penicillin, 100 μg/mL streptomycin Sigma-Aldrich/ Merck KGaA, Darmstadt, Germany), 2 mM glutamine Sigma-Aldrich/ Merck KGaA, Darmstadt, Germany) and 2% B27 supplement (Life Technologies, Carlsbad, CA, USA). Cells were then centrifuged, counted, and seeded onto poly-D-lysine-coated glass cover slides in 24-well plates for immunocytochemistry (80,000 cells/well). Seeded cells were maintained at 37 °C under a humidified atmosphere of 5% CO2 and 95% O2 in Neurobasal medium (Life Technologies, Carlsbad, CA, USA), and after 7 days in vitro (DIV), they were exposed to GD in order to induce αSyn aggregation [3 (link)]. GD was performed by incubating the cells with Phosphate Buffer (Sigma-Aldrich/ Merck KGaA, Darmstadt, Germany) supplemented with 2 mM glutamine and 2% of B27 for 1 h, at 37 °C. After GD, the cells were treated with different concentrations of I-threo and IV-threo for 24 h and fixed in 4% PFA (Bio-Optica, Milan, Italy) for 15 min, at 4 °C (n = 3 replicates).
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