The cell culture media (DMEM, MEM, RPMI 1640), 2-mercaptoethanol (ME), horse serum, sodium pyruvate, and phosphate-buffered saline (PBS) were purchased from Thermo Fisher Scientific. Fetal calf serum (FCS) for Caco-2 and HT29-MTX-E12, non-essential amino acids (NEAA), L-glutamine, D-glucose, penicillin/streptomycin (P/S), trypsin, phorbol 12-myristate 13-acetate (PMA), interferon-gamma (IFN-γ), lipopolysaccharides (LPS), Accutase, β-nicotinamide adenine dinucleotide sodium salt (NAD), lithium L-lactate, phenazine methosulfate (PMS), iodonitrotetrazolium chloride (INT), Triton X-100, bovine serum albumin (BSA), and 50 nm amine-modified polystyrene nanobeads (PS-NH2) were purchased from Sigma-Aldrich/Merck. Paraformaldehyde (PFA) was ordered from Carl Roth (Germany). The DQ12 quartz sample used in this study was kindly provided by the Institute of Occupational Medicine (IOM, Edinburgh, UK).
Paraformaldehyde pfa
Manufactured by Carl Roth
Sourced in Germany
4% paraformaldehyde (PFA) is a fixative solution commonly used in various laboratory applications. It serves as a preservative, maintaining the structural integrity of biological samples for further analysis or processing.
Automatically generated - may contain errors
Lab products found in correlation
D saccharose, by Carl Roth (2 mentions)
Dolethal, by Carl Roth (2 mentions)
Triton x 100, by Merck Group (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Merck Group (1 mentions)
Sodium pyruvate, by Thermo Fisher Scientific (1 mentions)
D glucose, by Merck Group (1 mentions)
Interferon gamma, by Merck Group (1 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Minimum essential medium (mem), by Thermo Fisher Scientific (1 mentions)
Trypsin, by Merck Group (1 mentions)
Horse serum, by Thermo Fisher Scientific (1 mentions)
2 mercaptoethanol, by Thermo Fisher Scientific (1 mentions)
Non essential amino acids (neaa), by Merck Group (1 mentions)
L glutamine, by Merck Group (1 mentions)
Accutase, by Merck Group (1 mentions)
Lipopolysaccharides (lps), by Merck Group (1 mentions)
Phenazine methosulfate, by Merck Group (1 mentions)
11 protocols using paraformaldehyde pfa
1
Cell culture and characterization protocol
2
Neonatal Mice Brain Development Study
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Animal experiments were performed according to protocols approved by the National Care Committee (Regierung Unterfranken; Wuerzburg, Germany) along with national and European laws on the protection of animals. Animals were provided with food and water ad libitum and housed with a 12 h light/12 h dark cycle. Pups were housed together with their dam to provide normal temperature and nutrition. A total of 128 C57BL/6NCrl neonatal mice (Charles River Laboratories; Sulzfeld, Germany) were block randomized at postnatal day 7 (P7) into the four treatment groups: (i) non-treated (NT; hypoxia, n = 16; normoxia, n = 16), (ii) vehicle-treated (VT; hypoxia, n = 16; normoxia, n = 16), (iii) rhGH-treated group (hypoxia, n = 16, normoxia, n = 16), and (iv) rhEPO-treated group (hypoxia, n = 16; normoxia, n = 16). After a regeneration period of 48 h (P9) or 7 d (P14), brains were dissected, snap-frozen in liquid nitrogen, and stored at −80 °C until required. The brains of a subgroup of pups (n = 3 per group) were perfused with Dulbecco’s phosphate-buffered saline (DPBS; pH 7.4) without Ca2+/Mg2+ (PAN-Biotech; Aidenbach, Germany) and fixed with 4% (w/v) paraformaldehyde (PFA; Carl Roth; Karlsruhe, Germany) for immunohistochemistry (IHC). Dissected brains were embedded in paraffin (Merck Millipore; Darmstadt, Germany). Supplementary Figure S4 presents the experimental design of this study.
3
Cuprizone-Induced Demyelination and Remyelination
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For myelination studies in vivo, eight-week-old male SV129, Tnc−/−, and Tnr−/− mice were fed 0.2% (w/w) cuprizone (Sigma-Aldrich; Cat. No. 370-81-0) mixed with powdered chow for six weeks to induce demyelination (Hillis et al., 2016 (link)), followed by a diet without cuprizone for one or two weeks (Albrecht et al., 2016) to allow for remyelination, as described previously (Ulc et al., 2019 (link)). Control mice received powdered chow without cuprizone. Four different experimental groups of mice were used. At the end of the experiment, mice of each group were perfused intracardially with 20 ml PBS in deep anesthesia (800 µl 0.9% (w/v) NaCl (Thermo Fisher Scientific; Cat. No. 7647-14-5), 50 µl Xylavet (xylazine) (10 mg/ml weight) (CP-Pharma, Handelsgesellschaft mbH; Cat. No. 1205), and 150 µl ketamine (150 mg/ml weight) (CP-Pharma, Handelsgesellschaft mbH; Cat. No. 1202)), and brains were removed and cut sagittally in two halves. Then, one hemisphere was fixed with 4% (w/v) paraformaldehyde (PFA) (Carl Roth; Cat. No. 4235.1) in PBS at 4°C for 48 h, before embedding in tissue-freezing medium for cryosectioning and following immunohistochemical staining. The second hemisphere was frozen in liquid nitrogen for RNA analysis.
4
Paclitaxel Cytotoxicity in Glial Cells
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Microglia, astrocytes and SGC were separately cultured in 24-well plates at 50,000/well in DMEM (10% FBS, 1% penicillin/streptomycin, 2 mM L-glutamine). Cells were cultured in white 96-well plates at a density of 12,000/well. After 24 h, cells were either not treated (control) or treated with 1 μM, 5 μM, 10 μM or 50 μM paclitaxel (obtained from University Hospital Cologne, Pharmacy) for 24 h before cell viability assay, NO- or ATP-assay, Enzyme-linked immunosorbent assay (ELISA), or RNA-extraction were performed (Lehmann et al., 2020 (link)). For immunocytochemical staining, cells were fixed with 4% paraformaldehyde (PFA; Carl Roth, Germany).
To obtain “conditioned medium” microglia, astrocytes and SGCs were either not stimulated (conditioned medium-control) or stimulated with paclitaxel 1 μM, 5 μM, 10 μM, or 50 μM for 24 h. After washing cells with 1x PBS, cells were incubated with fresh paclitaxel-free cell culture medium (DMEM containing 10% FBS, 1% penicillin/streptomycin, 2 mM L-glutamine) for 24 h. Conditioned medium containing the secretome but no paclitaxel was collected and stored at −20°C.
To obtain “conditioned medium” microglia, astrocytes and SGCs were either not stimulated (conditioned medium-control) or stimulated with paclitaxel 1 μM, 5 μM, 10 μM, or 50 μM for 24 h. After washing cells with 1x PBS, cells were incubated with fresh paclitaxel-free cell culture medium (DMEM containing 10% FBS, 1% penicillin/streptomycin, 2 mM L-glutamine) for 24 h. Conditioned medium containing the secretome but no paclitaxel was collected and stored at −20°C.
5
Multimodal Tissue Sampling for Neuroimmune Analysis
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Blood samples for flow cytometry were taken from the tail vein 1 day before sacrifice. Mice were sacrificed with CO2 before a non-perfused draining inguinal lymph node was collected for ELISPOT and blood was drawn from the inferior vena cava for ELISA analysis. Mice were then perfused with 4% paraformaldehyde (PFA) (Roth, Karlsruhe, Germany) in 0.01 M phosphate-buffered saline (PBS) (pH 7.4) for immunohistochemistry (IHC) or with 4% PFA/4% glutaraldehyde (GA) (Roth, Karlsruhe, Germany) in 0.01 M PBS (pH 7.4) for electron microscopy. The cerebellum, lumbar spinal cord, and the other draining inguinal lymph nodes were carefully dissected and post-fixed overnight at 4 °C under slight agitation. While samples for IHC were embedded in paraffin, samples for electron microscopy were embedded in Epon.
6
Cell Adhesion Dynamics on FN Substrates
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Cells were seeded on FN-AF488 substrates, incubated in DMEM containing 1% FBS at 37°C in 5% CO2 for 10 min to 24 h, and fixed with 4% paraformaldehyde (PFA) (Carl Roth, Karlsruhe, Germany) for 30 min. TIRF images were acquired using an iMIC microscope (FEI Life Sciences, Munich, Germany) and an APON 60× OTIRF oil immersion lens (Olympus, Hamburg, Germany). Epifluorescence images of FN in combined light microscopy/AFM experiments were obtained using an Achroplan 40×/0.80 W water-immersion lens (Zeiss, Oberkochen, Germany). For visualizing focal adhesions, cells were stained using a mouse monoclonal anti-vinculin antibody (V9131; Sigma-Aldrich, Taufkirchen, Germany).
7
FosB Immunostaining in Mouse Brain
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Twelve mice (VEH, n = 6; CORT, n = 6) randomly chosen from the behavioural group A were sacrificed 24 h following the attainment of their BP in the PR testing for FosB immunostaining. The animals were deeply anesthetised with an intraperitoneal injection of Dolethal (1 ml/kg, Vetoquinol), then transcardially perfused with 0.9% NaCl, followed by ice-cold 4% paraformaldehyde (PFA, Roth®, Karlsruhe, Germany), fixative in 0.1 M phosphate buffer pH 7.4. Once extracted, brains were postfixed overnight in the same fixative at 4°C and cryoprotected by immersion in a 15% sucrose solution (D(+)-Saccharose, Roth®, Karlsruhe, Germany) in a 0.1 M phosphate buffer 24 h at 4°C. The brains were then frozen by immersion in isopentane (2-methylbutane, Roth®, Karlsruhe, Germany) at −74°C using a Snap-FrostTM system (Excilone, France) and stored at −80°C until processed. The brains were cut in coronal 30-μm-thick serial sections, collected in a cryoprotector solution (1:1:2 glycerol/ethylene glycol/phosphate buffered saline – PBS; Roth®, Karlsruhe, Germany) and stored at −40°C.
8
LSFM Analysis of Mice Brain Vasculature
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For LSFM analyzes mice were sacrificed after four weeks of therapy on day 36. 2 h after injection of 20 µg VE-Cadherin-AlexaFluor647 intravenously, mice were anesthetized and transcardially perfused with 4% paraformaldehyde (PFA) (Carl Roth, Karlsruhe, Germany) and the brain was prepared and fixed with 4% PFA for 2 h. Samples were blocked with 2% FCS/PBS in 0.1% Triton-X (Carl Roth), incubated with propidium iodide (1 µg/ml, Merck) for 60 min at 4 °C. Organs became optically transparent after dehydration in a graded ethanol series (30–100% ethanol, each 1.5 h), followed by a clearing procedure including 2 h in 100% n-hexane (Merck) and 3 × 30 min clearing solution containing 1 part benzyl alcohol (Merck) and 2 parts benzyl benzoate (Merck). For 3D-imaging, we employed a homebuilt LSFM as previous described37 (link). The resulting multicolor stacks were processed and analyzed with Imaris Software 7.7.2 (Bitplane, Zurich, Switzerland). 3D reconstruction of vasculature, vasculature segmentation and analysis were performed with Imaris 8.1 software. Diameter, length as well as branch level were calculated for each dendrite of each vessel and the results were presented as histograms. Branch level was determined according to the new branching point and diameter change of segments. The branch level increased as soon as the segment diameter decreased in the branching point.
9
Spinal Cord TEM Sample Preparation
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All mice were sacrificed with CO2 seven days after the final treatment. For Simoa™ NfL analysis, blood was collected from the inferior vena cava, centrifuged at 1000× g for 10 min and the serum stored at −80 °C until analysis. Mice were perfusion-fixed with 4% paraformaldehyde (PFA; Roth, Karlsruhe, Germany) in PBS. The lumbar spinal cord was dissected and post-fixed at 4 °C overnight using transmission electron microscopy (TEM) fixing solution under slight agitation. The TEM fixing solution was prepared by dissolving 4 g 4% PFA and 2.14 g 0.1 M sodium cacodylate (Serva, Heidelberg, Germany) in 50 mL double-distilled water (ddH2O). After cooling and filtration, 16 mL 25% glutaraldehyde (Roth) and 17 mL 1.2% picric acid (AppliChem, Darmstadt, Germany) were added and the solution was filled with ddH2O to reach 100 mL. The pH was titrated with either hydrochloric acid (Roth) or sodium hydroxide (Roth) to reach a pH of 7.2—7.4. The samples were then washed with PBS for 24 h, changing the buffer three times.
10
Immunocytochemistry of Skin Fibroblasts
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Skin fibroblasts cell lines from the IP and control (C1, C2) were counted and seeded on coverslips (1.2 × 106 cells per 12 mm coverslip) by overnight incubation in 12-well plates [25 (link)]. Afterwards, cells were fixed for 20 min in 4% paraformaldehyde (PFA, Carl Roth, Karlsruhe, Germany) and blocked for 30 min in phosphate-buffered saline (PBS, Chemsolute, Renningen, Germany) with 0.1% Tween 20 (AppliChem, Darmstadt, Germany) and 5% bovine serum albumin (BSA, fraction V, Carl Roth). The ICC staining was performed with primary Anti-RBM10/S1-1 antibody (Abcam, Cambridge, UK, ab72423, 1:300) at room temperature for 2 h. Later, skin fibroblasts were incubated for 2 h at room temperature with secondary antibodies Alexa Fluor 568 (Thermo Fisher Scientific, A-10037, 1:2000). Each incubation was terminated by washing the cells in PBS supplemented with 0.2% Tween 20. Coverslips were mounted with DAPI Fluoromount-G (SouthernBiotech, Birmingham, AL, USA) and slides were analyzed with a fluorescence microscope (Axio Observer, Zeiss, Oberkochen, Germany) and Axiocam 512 mono (4248 × 2832 pixles). Microscope images were examined with ImageJ software (ImageJ, Bethesda, Maryland, USA).
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