Human brain tissue of 4 DLB (80 ± 7 years, post-mortem interval (PMI) 8 ± 3), 4 MSA (70 ± 7 years, PMI 8 ± 3 h), and 4 normal cases (74 ± 9 years; PMI, 14 ± 8 h) was obtained from the South Australian Brain Bank including five different anatomical regions: frontal cortex, temporal cortex, motor cortex, visual cortex and hippocampus. Formalin-fixed, paraffin-embedded sections (5 μm) underwent heat-induced antigen retrieval in 1 mM EDTA (pH 8, at 100 °C), followed by blocking with 20% normal horse serum (NHS). Primary rabbit anti-Munc18-1 (Sapphire Biosciences, Ab3451, 1:100) and mouse anti-α-syn (Abcam, LB509, 1:500) antibodies diluted in 1% NHS were incubated overnight, washed, then incubated with Alexa Fluor secondary antibodies (Invitrogen, A21202, A27034, A10042, all 1:200 in 1% NHS in TBS) in the dark for 90 min. Finally, slides were mounted in DAPI Pro-long gold (Life Technologies, Melbourne, Australia), and equivalent anatomical regions were imaged in diseased and control cases. Munc18 vesicles were counted in de-identified images as discrete puncta with Munc18 staining at least five-fold over background.
Ab3451
Manufactured by Abcam
Ab3451 is a primary antibody for use in immunoassays. It is a mouse monoclonal antibody that recognizes a specific antigen. The core function of this product is to bind to and detect the target antigen in samples.
Automatically generated - may contain errors
Lab products found in correlation
Irdye 680lt, by LI COR (2 mentions)
Sc 25778, by Santa Cruz Biotechnology (2 mentions)
Odyssey clx imager, by LI COR (2 mentions)
Protease inhibitor cocktail, by Roche (2 mentions)
A21202, by Thermo Fisher Scientific (1 mentions)
Prolong gold dapi, by Thermo Fisher Scientific (1 mentions)
Lb509, by Abcam (1 mentions)
A27034, by Thermo Fisher Scientific (1 mentions)
A10042, by Thermo Fisher Scientific (1 mentions)
Alexa fluor secondary antibody, by Thermo Fisher Scientific (1 mentions)
Image studio lite 5, by LI COR (1 mentions)
Image studio lite version 5, by LI COR (1 mentions)
Ab8224, by Abcam (1 mentions)
Ab3611, by Abcam (1 mentions)
Ab6671, by Abcam (1 mentions)
Ab64581, by Abcam (1 mentions)
Protein assay kit, by Bio-Rad (1 mentions)
5 protocols using ab3451
1
Munc18-1 Vesicle Quantification in Neurodegenerative Disorders
2
THP1 and RAW Cell Stimulation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For 0 (unstimulated), 2, 4, 6, 8 and ON, 100 ng/ml LPS and 100 ng/ml LTA were used to stimulate THP1 cells, whereas 1000 ng/ml LPS and 100 ng/ml LTA were used to stimulate RAW cells. Later, cells lysates were separated on an SDS-PAGE, transferred, and detected for STXBP1 (ab3451; Abcam) and GAPDH (10494-1-AP; Proteintech). See also Supplementary Fig. 2 .
3
Southern and Western Blot Analyses
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Southern and Western blot analyses were performed according standard protocols. For Southern blots, genomic DNA was extracted from ES cells and digested with BspHI for the 5’ probe or MfeI for the 3’ probe (Figure 1—figure supplement 1A ). 32P-labeled probes were used to detect DNA fragments. For Western blots, proteins were extracted from the brains at embryonic day 17.5 or 3 months of age using lysis buffer containing 50 mM Tris-HCl (pH 7.6), 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.5% Na-deoxycholate, 0.1% SDS, and 1 tablet of cOmplete, Mini, EDTA-free Protease Inhibitor Cocktail (Roche) in 10 ml buffer. Stxbp1 was detected by a rabbit antibody against the N terminal residues 58–70 (Abcam, catalog # ab3451, lot # GR79394-18, 1:2000 or 1:5000 dilution) or a rabbit antibody against the C terminal residues 580–594 (Synaptic Systems, catalog # 116002, lot # 116002/15, 1:2000 or 1:5000 dilution). Gapdh was detected by a rabbit antibody (Santa Cruz Biotechnology, catalog # sc-25778, lot # A0515, 1:300 or 1:1000 dilution). Primary antibodies were detected by a goat anti-rabbit antibody conjugated with IRDye 680LT (LI-COR Biosciences, catalog # 925–68021, lot # C40917-01, 1:20,000 dilution). Proteins were visualized and quantified using an Odyssey CLx Imager and Image Studio Lite 5.0 (LI-COR Biosciences).
4
Western Blot of Stxbp1 in Mouse Brain
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Western blot analyses were performed according to the protocols published previously (Chen et al., 2020) with minor modifications. Proteins were extracted from the entire mouse brains on postnatal day 0 or the cortices from 3-month-old mice. The lysis buffer contained 50 mM Tris-HCl (pH 7.6), 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.5% Na-deoxycholate, 0.1% SDS, and 1 tablet of cOmplete™, Mini, EDTA-free Protease Inhibitor Cocktail (Roche, catalog # SKU 11836170001) in 10 ml buffer. Stxbp1 was detected by a rabbit antibody against the N terminal residues 58-70 (Abcam, catalog # ab3451, lot #GR79394-18, 1:2,000 or 1:5,000 dilution) or a rabbit antibody against the C terminal residues 580-594 (Synaptic Systems, catalog # 116002, lot # 116002/15, 1:2,000 or 1:5,000 dilution). Gapdh was detected by a rabbit antibody (Santa Cruz Biotechnology, catalog # sc-25778, lot # A0515, 1:300 or 1:1,000 dilution). Primary antibodies were detected by a goat anti-rabbit antibody conjugated with IRDye 680LT (LI-COR Biosciences, catalog # 925-68021, lot # C40917-01, 1:20,000 dilution). Proteins were visualized and quantified using an Odyssey CLx Imager and Image Studio Lite version 5.0 (LI-COR Biosciences). Stxbp1 levels were normalized by the Gapdh levels. Each mouse was tested by both Stxbp1 antibodies and the results from the two antibodies were averaged.
5
Hippocampal Protein Extraction and Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mouse hippocampi were dissected into ice-cold phosphate-buffered saline (PBS) containing protease inhibitors and phosphatase inhibitors (Complete, Roche Diagnostics) and samples were ash-frozen in liquid nitrogen until further analysis. All the following procedures were then performed at 4 C. Frozen tissue was homogenised in ice-cold RIPA buffer (20 mM Tris (pH 7.4), 150 mM NaCl, 3 mM EDTA, and 1% Nonidet P-40) and 1 x protease inhibitor cocktail with a tissue homogenizer (IKA T10 basic ULTRA-TURRAX®). Total protein concentration was measured by the Bradford method (BioRad Protein assay kit). Primary antibodies were as following: complexin1/2, 1:2000, 122102 Synaptic Systems; Kv3.1, 1:2000, Alomone labs; synaptobrevin, 1:1000, ab77314 Abcam; synaptophysin, 1:1000, 5467, Cell Signaling; synapsin, 1:2000, ab64581, Abcam; SNAP-25, 1:1000, ab41726 Abcam; Munc 18, 1:1000, ab3451, Abcam; 3-NT, 1:5000, ab61342, Abcam; TPI, 1:1000, ab6671, Abcam; RAGE, 1:1000, ab3611, Abcam; ICSM35 1:1000, D-Gen Ltd; tubulin 1:5000, 2144, Cell Signaling; actin, 1:5000, ab8224, Abcam.
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!