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Whole-cell lysates were prepared in RIPA buffer (50mMTris-HCl pH 8.0, 150mMNaCl, 0.1% SDS, 0.5% Na-Deoxycholate, 1%IGEPAL CA-630) supplemented with PhosSTOP phosphatase inhibitors and cOmplete protease inhibitor cocktail (both Roche). BCA protein assay (ThermoScientific) was used for protein quantification. Samples were loaded on 4–15% precast polyacrylamide gels (Bio-Rad) and transferred to PVDF (Bio-Rad) membranes in Towbin buffer. Membranes were blocked in 5% bovine serum albumin (Applichem) and incubated overnight in primary antibodies phospho-p65 (1:1000; CST#3033), Caspase 8 (1:1000; CST#4790), cleaved Caspase 8 (1:1000, CST#8592), FLIP (1:1000, CST#56343) and Gapdh (1:3000; CST#14C10). IRDye800CW and 680RD donkey anti-rabbit secondary antibodies were used for detection at a 1:10,000 dilution (LI-COR). Protein bands were visualized with the ODYSSEY CLx imaging system (LI-COR).
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Samples were washed three times with PBS, fixed in formaldehyde (4% v/v in PBS) for 10 min and washed three times with PBS. For immunofluorescence labelling, fixed samples were incubated with primary antibodies in blocking buffer (3% w/v bovine serum albumin (#A6588, AppliChem) in PBS) for 1 h at room temperature. The samples were washed three times and stained with secondary antibodies in blocking buffer for 30 min at room temperature in the dark. For dual-colour labelling with secondary antibodies that had a cross-reactivity, antibodies were applied sequentially. Samples were briefly washed with PBS and then post-fixed with 4% v/v formaldehyde in PBS for 10 min, washed three times with PBS and stored at 4 °C in the dark.
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3

Immunocytochemical Analysis of Chondrogenic Differentiation

After 3 and 5 weeks of chondrogenic differentiation, cells were fixed in ice-cold methanol (POCH, Gliwice, Poland) and incubated at −20 °C. Later, the cells were washed and blocked for non-specific binding using 1% bovine serum albumin (BSA, AppliChem GmbH, Darmstadt, Germany) containing 0.5% Tween 20 (POCH, Gliwice, Poland) for 30 min. The cells were then incubated overnight at 4 °C with primary antibodies (Table S2). The next day, cells were washed 3 times in PBS containing 1% BSA and underwent 1h incubation with secondary antibodies at 37 °C (Table S2). Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich, Saint Louis, MO, USA) to show the nucleus. Images were taken using an Opta-Tech MW-100 microscope (Opta-Tech, Warsaw, Poland) under 200× magnification and documented using Opta-View software (Opta-Tech, Warsaw, Poland). The pictures were then merged and semiquantified as previously described [53 (link)] using an Image-J ver 1.50i (National Institute of Mental Health, Bethesda, MA, USA).
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Cultured neuronal cells infected with VSV-eGFP (MOI-0.01) were fixed with 4% (w/v) paraformaldehyde (PFA) in in 0.1 M phosphate buffer for 20 min, and incubated in blocking solution consisting of 0.2% (v/v) Triton X-100, 1% (w/v) bovine serum albumin (Applichem) and 10% goat serum in PBS. Primary antibody incubation was performed overnight in blocking solution without BSA using markers for neurons-(antiNeuN, Millipore, 1∶500) and astrocytes (anti-GFAP (Synaptic Systems, 1∶500). Secondary antibodies conjugated with Cy3 or Cy5 (Jackson Immunoresearch) were incubated at room temperature for 2 hours diluted 1∶500 in PBS. Images were acquired on LSM Carl Zeiss Confocal.
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CF41.Mg and MDCK cells (1×104/well) were seeded and grown on sterile glass coverslips, and then fixed with absolute methanol. Cells were washed and blocked with PBS plus 2% bovine serum albumin (Applichem GmbH, Darmstadt, Germany) for 30 min in a humidified chamber at room temperature. Subsequently, the cells were incubated with rabbit anti-COX-2 primary antibody clone SP21 (MA5-145; 1:50; Thermo Fisher Scientific, Inc.) at room temperature for 1 h as previously described (28 (link),29 (link)). Cells were then incubated with a goat anti-rabbit Alexa Fluor® 488 secary antibody (1:500; Invitrogen; Thermo Fisher Scientific, Inc.; A11008) for 1 h at room temperature. Coverslips were mounted using Vectashield® mounting media with DAPI (Vector Laboratories, Inc., Burlingame, CA, USA). Finally, the samples were analyzed with an epifluorescence microscope fitted with a color charge-coupled device camera.
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The
Leishmania-specific IgG isotype profile was determined with ELISA tests using SLA obtained from
L. majorpromastigotes. Three weeks after the termination of treatment,
plasma from all experimental mice was analyzed for the presence of IgG1- and IgG2a-specific antibodies. Plates (Sarstedt) were coated with 5 µg/mL of SLA diluted in carbonate buffer (15 mM
Na
2CO
3, 35 mM NaHCO
3), pH 9.6, and incubated overnight at 4 °C. Plates were blocked with 2% bovine serum albumin (AppliChem) and serial dilutions of plasma
samples (1/20 to 1/40 960) were added. Then, plates were incubated with biotinylated anti-mouse IgG1 (500 ng/mL) or IgG2a (250 ng/mL) (AbDSerotec), and HRP streptavidin solution (1/5000,
AbDSerotec) was added. Lastly, 3,3′,5,5′-tetramethylbenzidine substrate (ThermoFisher Scientific) was added, and absorbance was read at 450 nm. Results are expressed as antibody titer ± SD.
Antibody titer was determined as the maximum dilution in which the sampleʼs OD value was above the cutoff level (twice the mean value of blank wells).
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Cells were plated on poly‐L‐lysine (Sigma, cat. P1274)‐coated coverslips. Cells were fixed by 10‐min incubation in 4% paraformaldehyde (Alfa Aesar, 30525‐89‐4) at room temperature, permeabilized for 4 min in 1× PBS/0.5% Triton X‐100, washed with PBS, and blocked in 1% bovine serum albumin (Applichem, cat. A1391,0100) and 10% fetal bovine serum in PBS. Cells were incubated with primary antibody (Appendix Table S7) overnight at 4°C, followed by incubation with a fluorescent secondary antibody for 1 h at room temperature as previously described 90. Antibody solutions were made in PBS with 1% bovine serum albumin. Coverslips were mounted on glass slides using VECTASHIELD Antifade Mounting Medium with 49,6‐diamidino‐2‐phenylindole (DAPI) for DNA staining (Vektor, cat. H‐1200). For DNA repair experiments, cells were treated with 2 μΜ cisplatin for 6 h. For tissue stainings, tumors were fixed in 4% formaldehyde at 4°C, thoroughly washed in PBS, placed in 30% sucrose overnight, and frozen in optimal cutting temperature (OCT) compound (Tissue Tek, Sakura). Frozen 10 μm sections were obtained using a Leica (CM1950) cryostat. Sectioned tissues were washed three times in PBS, blocked for 1 h, and incubated with primary and secondary antibodies as described. Image processing and foci counts were performed using ImageJ.
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A total of 1.5 × 109 cells from an exponentially growing culture, or a
chemostat culture, was harvested in mid-exponential phase (OD600 ≈ 0.5) and
centrifuged for 4 min at 4,000 g and 4°C. The supernatant was discarded, the
cells washed or stained, and inoculated (∼2 × 107 cells
ml−1) into M9 medium plus acetate, succinate, fumarate, or malate. For washing,
the cells were resuspended in 5 ml ice-cold M9 medium, centrifuged a second time, and re-suspended
in 1 ml of room temperature M9 medium. For staining, the cells were resuspended in 500 μl of
room temperature dilution buffer C (Sigma-Aldrich). A freshly prepared mixture of 10 μl PKH26
or PKH67 dye (Sigma-Aldrich) and 500 μl dilution of buffer C (both at room temperature) was
added. After 3 min at room temperature, 4 ml ice-cold filtered M9 medium containing 1% (w/v)
bovine serum albumin (AppliChem) was added. After centrifugation, the supernatant was discarded and
the cells washed twice.
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MEFs were fixed in 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS; containing (in mM) 2.7 KCl, 1.5 KH2PO4, 137 NaCl, 8.1 Na2HPO4; pH 7.4) for 15 min at 4 °C, washed twice in PBS and stored at 4 °C in PBS containing 0.1% NaN3 until further processing. Permeabilization of MEFs were obtained by 1 h incubation at room temperature (RT) in blocking solution containing 0.3% Triton X-100 (Fluka, Buchs, Switzerland), 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES, pH 7.4, Sigma Aldrich, St. Loius, MO, USA) and 3% bovine serum albumin (AppliChem, Darmstadt, Germany). Thereafter, MEFs were incubated with primary antibodies either for 2 h at RT or overnight (o/n) at 4 °C, incubation time and temperature as well as dilution of primary antibodies is indicated below. After that, MEFs were washed twice in PBS and incubated for 1 h at RT with the appropriate secondary antibody (Alexa Fluor 488- or Alexa Fluor 546-conjugated goat anti-mouse IgG or goat anti-rabbit IgG; Invitrogen) diluted 1:1000 in blocking solution. To determine co-localization of proteins, we calculated the Pearson's correlation by using ImageJ plugin 'just another co-localization plugin' (National Institutes of Health) as described before.67
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The frozen section slides were dried for 1 h at 37 °C. After fixation with Roti-Histofix 4% (Carl Roth, Karlsruhe, Germany) for 15 min, the slides were blocked with 3% bovine serum albumin (AppliChem, Darmstadt, Germany) in PBS (Sigma/Merck, Darmstadt, Germany). Incubation with the anti-MUC1 (CellSignaling, Frankfurt am Main, Germany) antibody was performed overnight at 4 °C, followed by a 1 h incubation with the secondary rhodamine anti-mouse antibody (Dianova, Hamburg, Germany). Anti-EpCAM (Acris, Rockville, MD, USA) and anti-cytokeratin 8, 9, 18 and 19 (Abcam, Cambridge, UK) antibodies directly labeled with FITC were applied for 1 h at RT. Cell nuclei were visualized using Hoechst 33258. Images were taken using an inverted microscope at 20x magnification (Carl Zeiss Microscopy, Oberkochen, Germany).
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