The immunofluorescence staining was performed as previously described [35 (link)]. In brief equine stem cells were plated in a 96 well plate with a density of 10.000 cells per well. After 3 days when the cells had reached near confluency medium was aspirated from the cells and they were washed with PBS. After fixation with 4% paraformaldehyde for 20 min at room temperature MSCs were incubated over night with primary antibodies. The antibodies used in this study include the following: anti CD9 (monoclonal mouse, HI9a, BioLegend, 1:1000), anti CD63 (monoclonal mouse, MX-49.129.5, Santa Cruz, 1:500) and anti CD81 (monoclonal mouse, sc-166,029, Santa Cruz, 1:500). For negative controls the primary antibody was replaced by non-immune serum (rat anti mouse IgG isotypes, Invitrogen, 1:200). Then a secondary goat anti mouse Cy3 antibody (Dianova, Hamburg, Germany, 1:150) was applied for 1 h. Counterstaining was performed by using Hoechst-dye (bisBenzimide H33258, Sigma-Aldrich, Steinheim, Germany).
Mx 49
Manufactured by Santa Cruz Biotechnology
Sourced in United States
The MX-49.129.5 is a compact and versatile piece of laboratory equipment designed for a variety of applications. It functions as a high-performance centrifuge capable of separating samples at controlled speeds and temperatures. The product specifications and technical details are available upon request.
Automatically generated - may contain errors
Lab products found in correlation
Sc 166029, by Santa Cruz Biotechnology (1 mentions)
Csaii kit, by Agilent Technologies (1 mentions)
Alexa 488, by Thermo Fisher Scientific (1 mentions)
W6b3c1, by Miltenyi Biotec (1 mentions)
Tsa plus system, by PerkinElmer (1 mentions)
Epr2949, by Santa Cruz Biotechnology (1 mentions)
Molecular imager, by Bio-Rad (1 mentions)
Trs buffer, by Agilent Technologies (1 mentions)
Sc 7309, by Santa Cruz Biotechnology (1 mentions)
Sc 74497, by Santa Cruz Biotechnology (1 mentions)
Alexa fluor 488 donkey anti rabbit igg a21206, by Thermo Fisher Scientific (1 mentions)
6 protocols using mx 49
1
Immunofluorescence Staining of Equine Stem Cells
2
Immunofluorescence of TIMP-1 and Stem Cell Markers
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HIER was performed in TEG buffer (10 mmol/L Trisbase and 0.5 mmol/L EGTA) followed by incubation for 60 min with a TIMP-1 (VT7 [54 (link)], 1 + 4000) or a CD133 (W6B3C1, 1 + 40, Miltenyi, Belgium) antibody. The primary antibody was detected using the CSA II kit (Dako) (TIMP-1 and CD133) or TSA Plus System (Perkin Elmer) (TIMP-1). HIER was repeated followed by incubation for 60 min with an antibody against Sox2 (245,610, 1 + 800, R&D Systems, USA), CD63 (MX-49.129.5, 1 + 800, Santa Cruz), or TIMP-1 (VT7, 1 + 4000). A secondary antibody with Alexa488 (Invitrogen, USA) was used for Sox2, whereas TSA (Perkin Elmer) was used for TIMP-1 and CD63. Nuclei were counterstained with DAPI. The double-immunofluorescence stainings were analysed using a Nikon Eclipse TE2000-E inverted confocal microscope.
3
Exosomal Protein Characterization by Western Blot
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After isolation, we lysed the exosomes in lithium dodecyl sulphate buffer (LDS buffer) and measured the protein concentration by BCA protein assay kit. Later, protein extracts were separated on SDS-PAGE, transferred to a PVDF membrane, and blocked with 5% milk in PBS and incubated overnight at 4°C with primary antibodies for CD9 (Abcam; EPR2949, 1:2000), CD63 (Santa Cruz Biotechnology; MX-49.129.5, 1:200), and HSP70 (Santa Cruz Biotechnology; sc-137210, 1:200). After washing, the membranes were incubated with the horseradish peroxidase-conjugated secondary antibody (Nanjing Jiancheng, 1:1000) for 1 h and washed again. Lastly, membranes were developed and images were collected on Bio-Rad Molecular Imager.
4
Immunohistochemical Evaluation of Glioma Markers
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Formaldehyde-fixed, paraffin-embedded tissue was cut into three μm sections on a microtome and stained routinely with haematoxylin-eosin to define representative tumor regions. For immunohistochemical staining with CD63, paraffin sections were then deparaffinized, and endogenous peroxidase activity was quenched followed by heat-induced epitope retrieval (HIER) in TRS buffer (Dako, Denmark). The sections were subsequently incubated for 60 min with a CD63 monoclonal antibody (MX-49.129.5, Santa Cruz, 1 + 1000, USA). The antigen-antibody complex was detected using anti-mouse EnVision (Dako) and visualised with diaminobenzidine (DAB) as chromogen. Finally, the sections were counterstained with Mayer’s haematoxylin.
Immunohistochemical staining for mutation in isocitrate dehydrogenase 1 (IDH-1) was performed as previously described [51 (link), 52 (link)]. For diffuse and anaplastic astrocytomas that were IDH-1 wildtype by immunohistochemistry, next generation sequencing was performed using the 20-gene panel as reported by Zacher et al. [53 (link)].
Immunohistochemical staining for mutation in isocitrate dehydrogenase 1 (IDH-1) was performed as previously described [51 (link), 52 (link)]. For diffuse and anaplastic astrocytomas that were IDH-1 wildtype by immunohistochemistry, next generation sequencing was performed using the 20-gene panel as reported by Zacher et al. [53 (link)].
5
Antibodies for Cellular Protein Analysis
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Primary antibodies used in the research include mouse monoclonal anti-CD36 (sc-7309, SantaCruz, Dallas, TX, USA; WB: 1:1000, IC: 1:100), mouse monoclonal anti-CD63 (MX-49.129.5, SantaCruz; WB: 1:500), mouse monoclonal anti-GAPDH (G8795, Sigma; WB: 1:2000), rabbit polyclonal anti-GLUT1 (PA5-16793, Thermo Fisher Scientific®; WB: 1:1000, IC: 1:100, IF: 1:100), mouse monoclonal anti-GLUT4 (2213; Cell Signaling, Danvers, MA, USA; WB 1:1000), rabbit polyclonal anti-LC3B (2775, Cell Signaling; WB: 1:1000), mouse monoclonal anti-GLUT3 (sc-74497, SantaCruz; WB: 1:500) rabbit polyclonal anti-PARP (9542, Cell Signaling; WB: 1:1000), mouse monoclonal anti-p21 (2946, Cell Signaling; WB: 1:2000), rabbit polyclonal anti- ERVW-1 (SAB2108833, Sigma; WB: 1:1000). HRP-conjugated secondary antibodies were obtained from Bio-Rad (Bio-Rad.) and used at a concentration of 1:3000. For IF, Alexa Fluor® 488 donkey anti-rabbit IgG (A21206) were purchased from ThermoFisher Scientific® (ThermoFisher Scientific®).
6
CD63 Immunohistochemistry in Tumor Samples
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Haematoxylin and eosin-stained slides were reviewed,andacore(3mmindiameter)ofthemost representative tumour area was prepared from each formalin-fixed paraffin-embedded block. Immuno-histochemicalstainingwasperformedon4-μm-thick sections obtained from tissue microarray blocks. Tissue sections were stained with a monoclonal anti-CD63 antibody at a dilution of 1 : 500 (MX-49.129.5, Santa Cruz Biotechnology, CA, USA) using an automated immunostainer (Benchmark Ultra, Ventana Medical Systems Inc., Tucson, AZ, USA). Tumour-infiltrating immune cells were used asthepositivecontrol,andtheprimaryantibodywas omittedforthenegativecontrol.
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