Mice were euthanized with CO2 and perfused with PBS followed by 4% paraformaldehyde (PFA, pH=7.4). Mouse brains were extracted, post-fixed overnight at 4°C in 4% PFA and coronally sectioned at 100 μm intervals on a vibratome (Leica, VT-1000s). Brain sections were blocked (10% donkey serum, 0.2% Triton X-100 in PBS) for an hour at room temperature followed by overnight primary antibody incubation in block buffer at 4°C. The following primary antibodies were used: rabbit anti-c-Fos (1:500, Cell Signaling, #2250), sheep anti-FOXP2 (1:2000, R&D Systems, AF5647), rabbit-anti-ETV1 (1:500, Abcam, ab81086), chicken anti-GFP (1:1000, Abcam, ab13970), rat anti-mCherry (1:500, Invitrogen, 16D7). After washing 3 times with PBS, the brain sections were stained with secondary antibodies (1:500, Jackson Immunoresearch) and DAPI (2 μg/ml) for 4 hours at room temperature. After another 3 PBS washes, sections were mounted on glass slides and imaged on a confocal microscope (TCS SP8, Leica, using Leica Application Suite X 3.5.5.19976).
Ab81086
Manufactured by Abcam
Sourced in United States
Ab81086 is a recombinant anti-PACS1 antibody produced in rabbit. It is designed for use in Western blotting, immunohistochemistry, and immunocytochemistry applications.
Automatically generated - may contain errors
Lab products found in correlation
Vt1000, by Leica (4 mentions)
Ab13970, by Thermo Fisher Scientific (2 mentions)
Af5647, by Abcam (2 mentions)
Rat anti mcherry, by Thermo Fisher Scientific (2 mentions)
Application suite x 3, by Leica (2 mentions)
Tcs sp8, by Leica (2 mentions)
Ab53554, by Santa Cruz Biotechnology (2 mentions)
Ab5683, by Santa Cruz Biotechnology (2 mentions)
Ab72428, by Abcam (2 mentions)
Fluorophore conjugated secondary antibody, by Jackson ImmunoResearch (2 mentions)
Sc 52g, by Abcam (2 mentions)
Sc 648, by Abcam (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)
Fv1000, by Olympus (2 mentions)
Ab13970, by Abcam (2 mentions)
Ab31419, by Abcam (1 mentions)
Dynabeads, by Abcam (1 mentions)
Ab4729, by Abcam (1 mentions)
Hiseq 2500, by Illumina (1 mentions)
Truseq chip sample preparation protocol, by Illumina (1 mentions)
8 protocols using ab81086
1
Immunohistochemical Profiling of Mouse Brain
2
Immunohistochemical Analysis of Mouse Brain
3
Immunohistochemical Analysis of Mouse Brain
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Animals were sacrificed with ketamine and xylazine, and perfused with 10 ml of PBS followed by 10 ml of 4% PFA in PBS (pH 7.4). Brains were dissected and post-fixed overnight in 4% PFA in PBS. 100 μm coronal brain sections were prepared using a vibratome (VT-1000S, Leica). After blocking with 10% FBS/0.2% Triton X-100 in PBS in the presence of 0.2% Triton X-100 for 1 hr, sections were incubated with primary antibodies overnight at 4C. The primary antibodies (1:500 dilution) used in these studies were: rabbit anti-CamKII (Abcam, ab5683), goat anti-c-Fos (Santa Cruz, SC-52G), goat anti-GFAP (Abcam, ab53554), rabbit anti-nNOS (Santa Cruz, sc-648), goat anti-nNOS (abcam ab72428), rabbit anti-ETV-1 (Abcam, ab81086), and chicken anti-GFP (Abcam, ab13970). Sections were washed twice with PBS, followed by a >3 hr incubation with fluorophore-conjugated secondary antibodies (1:500 dilution, Jackson Immuno Research). Fluorescent images were acquired and processed using a confocal microscope (FV1000, Olympus). In some experiments, brain sections were counterstained with DAPI (Sigma Aldrich).
4
In Situ Localization of miR-17-5p and ETV1 in TNBC
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The localization of miR-17-5p and ETV1 was observed by in situ hybridization (ISH) and immunohistochemistry (IHC), respectively. In situ observation of miR-17-5p was performed using 4-μm sections of samples with a digoxigenin-labelled oligonucleotide miR-17-5p detection probe (Exiqon, USA), as previously described [13 (link)]. miR-17-5p probe sequence was (5′-3′) CTACCTGCACTGTAAGCACTTTG. Immunohistochemical staining of ETV1 was performed according to the manufacturer’s protocol on paired TNBC and adjacent normal tissues. The negative control was performed by replacing the primary antibody with preimmune rabbit serum. The immunoreactivity score method based on the proportion and intensity of positively stained tumor cells was employed according to our previous description [6 (link)].
The following antibodies were used for immunohistochemical staining and western blotting in this study: anti-GAPDH (ab181602) ) and ab81086.html">anti-ETV1 antibodies (ab81086) were purchased from Abcam (USA). Appropriate secondary antibodies were obtained from Cell Signaling Technology (USA).
The following antibodies were used for immunohistochemical staining and western blotting in this study: anti-GAPDH (
5
ChIP-qPCR and ChIP-seq Analysis of Transcription Factor Binding
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
ChIP-qPCR and ChIP-seq were carried out as described previously [13 (link)]. For ChIP-qPCR for AP1, a JUN antibody (Abcam (ab31419)) was used and for histone acetylation a H3K27ac antibody (Abcam (ab4729)) was used. For ChIP-seq, 3x107 cells, 3 μg antibody (ETV1; Abcam (ab81086)) and 30 μl Dynabeads were used per experiment. Parallel control experiments were run with ChIP-qPCR using rabbit IgG; Millipore (12–370). Library preparation was performed using the TruSeq ChIP Sample Preparation Protocol (Illumina) and DNA libraries were sequenced using the HiSeq 2500 (Illumina).
Sequencing tags/reads from the ETV1 ChIP-seq experiment in OE33 cells were aligned to the NBCI Build hg19 of the human genome with Bowtie v2.2.3 [38 (link)]. Up to two mismatches were allowed. Only reads with a mapping quality >q30 were retained. Peak calling was performed on individual replicates and merged datasets with MACS v2.1.0 software [39 (link)] using default parameters. Data are deposited in ArrayExpress (Accession number: E-MTAB-5168)
Sequencing tags/reads from the ETV1 ChIP-seq experiment in OE33 cells were aligned to the NBCI Build hg19 of the human genome with Bowtie v2.2.3 [38 (link)]. Up to two mismatches were allowed. Only reads with a mapping quality >q30 were retained. Peak calling was performed on individual replicates and merged datasets with MACS v2.1.0 software [39 (link)] using default parameters. Data are deposited in ArrayExpress (Accession number: E-MTAB-5168)
6
Chromatin Immunoprecipitation Protocol for HCT116 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Chromatin immunoprecipitation assays were performed on formaldehyde-treated HCT116 cells essentially as previously described (73 (link)). ETV1 and JMJD1A were precipitated with the rabbit polyclonal antibodies, ab81086 (Abcam) and NB100-77282 (Novus Biologicals), respectively, while control rabbit IgG was purchased from Santa Cruz Biotechnology, Inc. (sc-2027). Approximately 2 µg of the antibodies were employed for the immunoprecipitations that were performed at 4°C over-night (74 (link)). Genomic DNA was amplified in two steps with nested primers (75 (link)). The first PCR encompassed the following temperature steps: 97°C for 1 min; 9 cycles of 97°C for 20 sec, 65°C (-1°C per cycle) for 20 sec, 72°C for 40 sec; 20 cycles of 97°C for 20 sec, 56°C for 20 sec, 72°C for 40 sec; 72°C for 4 min followed by cooling down to 4°C. The second PCR was performed in the same manner except that 31 instead of 20 cycles were used. The primer sequences are presented in Table SII .
7
Immunohistochemical Profiling of Mouse Brain
8
Protein Detection by Western Blotting
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Tissue samples were homogenized, and proteins were detected using Western blotting [24] .
Briefly, total proteins was separated by SDS-PAGE and transferred to the nitrocellulose membrane. Blots were blocked and then incubated with primer antibodies including anti-actin
(1:10000, Abcam A2066), anti-PEA3 (1:500, Abcam, ab101455), anti-ERM (1:1000, Abcam, ab102010) and anti-ER81 (1:1000, Abcam, ab81086). After washing, HRP conjugated secondary antibodies were applied for 1 hr at room temperature before applying ECL solution (Amersham Biosciences), exposed to film for 10-20 min, and developed by hand. Protein bands were quantified using GeneSnap acquisition and GeneTools analysis software.
Briefly, total proteins was separated by SDS-PAGE and transferred to the nitrocellulose membrane. Blots were blocked and then incubated with primer antibodies including anti-actin
(1:10000, Abcam A2066), anti-PEA3 (1:500, Abcam, ab101455), anti-ERM (1:1000, Abcam, ab102010) and anti-ER81 (1:1000, Abcam, ab81086). After washing, HRP conjugated secondary antibodies were applied for 1 hr at room temperature before applying ECL solution (Amersham Biosciences), exposed to film for 10-20 min, and developed by hand. Protein bands were quantified using GeneSnap acquisition and GeneTools analysis software.
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!