NTA analysis of milk sEV from BLV-infected and uninfected cattle was performed using a NanoSight LM10V-HS, NTA 3.4 instrument by an assigning company (Quantum Design Japan, Tokyo, Japan). Morphological examination of isolated milk sEV from BLV-infected and uninfected cattle was carried out by TEM as described in a previous study41 (link) with slight modifications. The sEV pellet was diluted 10 times from its original concentration with distilled water, applied to glow-discharged carbon support films on copper grids followed by stained with 2% uranyl acetate. The samples were then visualized under an electron microscope, JEM-2100F (JEOL, Tokyo, Japan) at 200 kV. WB analysis was carried out as described in a previous study41 (link) with slight modifications. The primary antibodies, anti-CD63 (1:400, M-13, SC-31214, Santa Cruz Biotechnology, Santa Cruz, CA, USA) or anti-HSP70 (1:100, N27F3-4, Enzo Life Science, Farmingdale, NY, USA) following the secondary antibodies, anti-goat IgG donkey antibody (1:2000, SC-3851, Santa Cruz Biotechnology) or anti-mouse IgG sheep antibody (GE Healthcare) conjugated with horseradish peroxidase, were used as described above.
Sc 3851
Manufactured by Santa Cruz Biotechnology
Sourced in United States
SC-3851 is a laboratory equipment product manufactured by Santa Cruz Biotechnology. It is designed for general laboratory use, but its core function and detailed specifications are not available in this unbiased, factual description.
Automatically generated - may contain errors
Lab products found in correlation
Jem 2100f, by JEOL (1 mentions)
Dako envision system, by Agilent Technologies (1 mentions)
Trypsin, by Merck Group (1 mentions)
Af2948, by R&D Systems (1 mentions)
Ecl western blotting detection system, by GE Healthcare (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Na9310v, by GE Healthcare (1 mentions)
Protein a sepharose, by GE Healthcare (1 mentions)
Las 4000 mini, by Fujifilm (1 mentions)
3 protocols using sc 3851
1
Characterization of Milk-Derived Small Extracellular Vesicles
2
Evaluation of Cell Proliferation, Inflammation, and Adhesion
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To evaluate proliferation, inflammatory response, and cell adhesion, immunohistochemistry was performed. Proliferating nuclear antigen (PCNA, clone PC10, mouse IgG, Chemi-Con, Billerica, MA, USA) served as a marker for proliferation, tumor necrosis factor alpha (TNF-alpha, clone M-18, goat IgG, Santa Cruz Biotechnology, Santa Cruz, CA, USA) for inflammation and Fibronectin (FBN, clone C-20, goat IgG Santa Cruz Biotechnology, Santa Cruz, CA, USA) for cell–matrix interactions. After dissolving of Technovit 9100 NEU and antigen retrieval with trypsin (Sigma Aldrich, St. Louis, MO, USA), the antibodies were applied for 10 h. For mouse antibody detection, the Dako envision system (Dako, Glostrup, Denmark) was used according to the manufacturer’s protocols. As secondary antibody to the TNF-alpha and FBN staining, an anti-goat antibody (Sc 3851; Santa Cruz Biotechnology, Santa Cruz, CA, USA) was applied.
3
Quantifying Amyloid Fibril Protein Levels
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Amyloid fibrils were electrophoresed on 12% sodium dodecyl sulfate polyacrylamide gels.
After electrophoresis, gels were stained with Coomassie Brilliant Blue (CBB). Another set
of electrophoresed gels was transferred onto PVDF membranes (Millipore, Billerica, MA,
USA) and blocked with 5% skim milk in phosphate buffered saline (PBS) supplemented with
0.1% Tween 20 (PBST). The membranes were incubated with anti-mouse SAA1 goat antibody
(1:5,000) (AF2948, R&D Systems, Minneapolis, MN, USA), anti-bovine SAA1 monoclonal
antibody, 25BF12 (1:1,600) [29 (link)], which was purified
from the culture supernatant of hybridoma cells by protein A-sepharose (GE Healthcare), or
anti-chicken SAA1 mouse antibody (1:1,600) [22 (link)] in
1% skim milk in PBST at room temperature for 1 h. Membranes were washed three times with
PBST and incubated with anti-goat IgG donkey antibody F (ab’)2 conjugated with
horseradish peroxidase (HRP) (1:1,000) (SC3851, Santa Cruz Biotechnology, Dallas, TX,
USA), anti-mouse IgG F (ab’)2 conjugated with HRP (1:1,600)
(NA9310V, GE Healthcare, Buckinghamshire, UK) in 1% skim milk in PBST at room temperature
for 45 min. The membranes were washed four times with PBST. The band of amyloid fibrils
was detected by an ECL Western Blotting Detection System (GE Healthcare), and bands in
Western blot analysis were visualized by an LAS 4000 mini (Fujifilm, Tokyo, Japan).
After electrophoresis, gels were stained with Coomassie Brilliant Blue (CBB). Another set
of electrophoresed gels was transferred onto PVDF membranes (Millipore, Billerica, MA,
USA) and blocked with 5% skim milk in phosphate buffered saline (PBS) supplemented with
0.1% Tween 20 (PBST). The membranes were incubated with anti-mouse SAA1 goat antibody
(1:5,000) (AF2948, R&D Systems, Minneapolis, MN, USA), anti-bovine SAA1 monoclonal
antibody, 25BF12 (1:1,600) [29 (link)], which was purified
from the culture supernatant of hybridoma cells by protein A-sepharose (GE Healthcare), or
anti-chicken SAA1 mouse antibody (1:1,600) [22 (link)] in
1% skim milk in PBST at room temperature for 1 h. Membranes were washed three times with
PBST and incubated with anti-goat IgG donkey antibody F (ab’)2 conjugated with
horseradish peroxidase (HRP) (1:1,000) (SC3851, Santa Cruz Biotechnology, Dallas, TX,
USA), anti-mouse IgG F (ab’)2 conjugated with HRP (1:1,600)
(NA9310V, GE Healthcare, Buckinghamshire, UK) in 1% skim milk in PBST at room temperature
for 45 min. The membranes were washed four times with PBST. The band of amyloid fibrils
was detected by an ECL Western Blotting Detection System (GE Healthcare), and bands in
Western blot analysis were visualized by an LAS 4000 mini (Fujifilm, Tokyo, Japan).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!