Immunofluorescent staining was performed as described previously (Gao et al., 2019 (link)). Tissue sections were blocked for 1 hr at room temperature in PBST (0.3% Triton X-100) with 5% normal donkey serum, followed by incubation with primary antibodies at 4°C overnight and secondary antibodies at room temperature for 2 hr in PBST (0.3% Triton X-100) with 1% normal donkey serum. Primary antibodies used for immunofluorescent staining were anti-MOR (rabbit, 1:500 for brain and spinal cord, 1:1000 for DRG, ABCAM, ab134054), anti-CGRP (goat, 1:500, ABCAM, ab36001), FITC-IB4 (1:200, Sigma, L2895), anti-c-Fos (rabbit, 1:15000, Synaptic System, 226003), anti-VGluT2 (guinea pig, 1:2000, Millipore, AB2251-I), anti-GAD67 (mouse, 1:500, Millipore, MAB5406). Secondary antibodies were donkey anti-goat IgG-Cy5, donkey anti-rabbit IgG-Cy3, donkey anti-rabbit IgG-Alexa 488, donkey anti-mouse IgG-Alexa 488 and donkey anti-guinea pig IgG-Cy5 (1:200, Jackson ImmunoResearch Laboratories).
Ab134054
Manufactured by Abcam
Sourced in United Kingdom
Ab134054 is a recombinant rabbit monoclonal antibody that recognizes an epitope within the C-terminus of human Smad4. It is suitable for use in various immunoassays, including Western blotting, immunohistochemistry, and immunocytochemistry.
Automatically generated - may contain errors
Lab products found in correlation
Cy3 donkey anti rabbit igg, by Jackson ImmunoResearch (1 mentions)
Donkey anti mouse igg alexa 488, by Jackson ImmunoResearch (1 mentions)
Ib4 fitc, by Merck Group (1 mentions)
Mab5406, by Merck Group (1 mentions)
Ab2251 1, by Merck Group (1 mentions)
Donkey anti rabbit igg alexa488, by Jackson ImmunoResearch (1 mentions)
Ab76003, by Abcam (1 mentions)
Ab218106, by Abcam (1 mentions)
Ab8226, by Abcam (1 mentions)
Ds u3, by Nikon (1 mentions)
A8020, by Solarbio (1 mentions)
Protein assay kit, by Beyotime (1 mentions)
Ecl system, by Thermo Fisher Scientific (1 mentions)
E cadherin, by Abcam (1 mentions)
N cadherin, by Abcam (1 mentions)
Ab16051, by Abcam (1 mentions)
Twist, by Abcam (1 mentions)
Gapdh 5174, by Cell Signaling Technology (1 mentions)
Pe10 catheter, by Smith & Nephew (1 mentions)
Ab12169, by Abcam (1 mentions)
8 protocols using ab134054
1
Immunofluorescent Staining Protocol for Neuronal Markers
2
Protein Expression Analysis by Western Blot
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Western blot analysis was performed as described previously (13 (link)). Primary antibodies include OGFR (Abcam, ab1717), μ-opioids receptor (Abcam, ab134054), uPA (Abcam,ab218106), MMP-9 (Abcam, ab76003). Anti-β-actin (Abcam, ab8226) was used as an internal control. Immune complexes were visualized using the Beyo ECL Plus.
3
Triple Labeling of Rabbit IgG Targets in Brain Sections
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A rabbit IgG labeling kit (Zenon Tricolor Rabbit IgG labeling Kit #1; Molecular Probes, Eugene, OR) was used for triple-labeling with the rabbit polyclonal IgGs against: σ1R (internal region 139–157, Invitrogen, Madrid, Spain, 42-3300; Alexa Fluor 488), NMDAR NR1 subunit (C-terminus, Merck-Millipore, Chemicon AB9864; Alexa Fluor 555), and MOR (C-terminal region, Abcam, Cambridge, United Kingdom, ab134054; Alexa Fluor 647). Mouse coronal brain sections with the PAG were incubated with the labeling complex overnight and examined by confocal microscopy (Leica TCS SP-5/LAS AF Lite Software; Microsystems, GmbH, Hohenstein-Ernstthal, Germany). Controls for immunohistochemistry were performed according to standard protocols (for further see details in Supplementary Materials and Methods section and Supplementary Fig. S3 ).
4
Immunohistochemical Analysis of Opioid Receptor
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Immunohistochemistry was performed according to the standard protocols. The
sample that had been fixed and sliced was soaked in 4% paraformaldehyde
(phosphate-buffered saline [PBS]) for 24 hours. In order to eliminate enzymatic
activity, tissues were incubated in 3% H2O2 for 10 minutes
and washed 3 times for 5 minutes using PBS. The samples were blocked using 5%
bovine serum albumin (A8020, Solarbio, diluted in PBS) and incubated at room
temperature for 30 minutes. The blocking serum was removed, primary antibody was
added (anti-MOR antibody, 1:50, ab134054, Abcam, Cambridge, United Kingdom), and
then incubated at 4°C overnight. After washing with PBS twice and followed by
reacting with the secondary immunoglobulin at 37°C for 30 minutes, direct
observation with an inverted microscope(Nikon Ci-S) and analysis using
a Nikon imaging system (Nikon DS-U3) were performed.
Immunohistochemical images of tumor tissue were collected, and 200-fold images
were taken for semiquantitative analysis. The cells with opioid receptor
peptides staining specificity of the secondary antibody, the color of positive
cells was deeper than others. Five sections of each group were randomly
selected, and each of the slides was taken from the top left, right top, middle,
bottom left, and right bottom fields for counting. Each field was counted by 2
different researchers.
sample that had been fixed and sliced was soaked in 4% paraformaldehyde
(phosphate-buffered saline [PBS]) for 24 hours. In order to eliminate enzymatic
activity, tissues were incubated in 3% H2O2 for 10 minutes
and washed 3 times for 5 minutes using PBS. The samples were blocked using 5%
bovine serum albumin (A8020, Solarbio, diluted in PBS) and incubated at room
temperature for 30 minutes. The blocking serum was removed, primary antibody was
added (anti-MOR antibody, 1:50, ab134054, Abcam, Cambridge, United Kingdom), and
then incubated at 4°C overnight. After washing with PBS twice and followed by
reacting with the secondary immunoglobulin at 37°C for 30 minutes, direct
observation with an inverted microscope
a Nikon imaging system (Nikon DS-U3) were performed.
Immunohistochemical images of tumor tissue were collected, and 200-fold images
were taken for semiquantitative analysis. The cells with opioid receptor
peptides staining specificity of the secondary antibody, the color of positive
cells was deeper than others. Five sections of each group were randomly
selected, and each of the slides was taken from the top left, right top, middle,
bottom left, and right bottom fields for counting. Each field was counted by 2
different researchers.
5
Western Blot Analysis of Protein Markers
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Cells were lysed and protein concentration was quantified using a protein assay kit (Beyotime). Then, protein samples were separated by SDS-PAGE and transferred to a polyvinylidene fluoride (PVDF) membrane. After being blocked with 5% milk at room temperature for 1 h, the membrane was incubated overnight at 4°C with the following antibodies as indicated: MOR (ab134054; Abcam), AKT (4685S; CST), phospho-AKT (4060S; CST), β-catenin (ab16051; Abcam), N-cadherin (ab18203; Abcam), Twist (ab18203; Abcam), E-cadherin (ab76055; Abcam) and β-actin (4970s; CST). Then, HRP-conjugated secondary antibody (KPL) was added and incubated at room temperature for 2 h. Finally, protein bands were visualized using ECL system (Thermo Fisher), and quantified with a chemiluminescence gel imaging system (Tanon 5200; Tanon, Beijing China).
6
Validating Specificity of Anti-MOR1 Antibody
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The main series of experiments was performed with the rabbit monoclonal anti-MOR1 antibody raised against the 350–450 amino acid sequence of the human MOR1 C-terminal (ab134054, Abcam, MA, USA). To test for false-positive reactions, we confirmed the specificity of the immunostaining in blocking experiments using MOR peptide (ab239748, Abcam, MA, USA). The anti-MOR1 antibody (×500) was preincubated with MOR peptide (0.5 or 0.05 μg/ml) for 15 h in 3% bovine serum albumin (BSA) buffer before immunostaining. These preabsorbed solutions of primary antibody were used for immunostaining in striatal sections from WT mice as described below. The positive control was the section incubated with anti-MOR1 antibody (×500) in 3% BSA, and the negative control was the section incubated with 3% BSA.
7
Walker 256 Rat Ascites Carcinoma Cell Line Protocol
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The cell line Walker 256 rat ascites carcinoma cell line was obtained from
American Type Culture Collection (Manassas, Virginia) The μ-opioid receptor
rabbit monoclonal antibody (ab134054) was purchased from Abcam (Cambridge,
Massachusetts). The GAPDH [5174] was purchased from Cell Signaling Technology
(CST, Danvers, Massachusetts). The PE10 catheter was purchased from Smiths
Medical (Ashford, Kent, United Kingdom). The Von Frey fiber probe (NC12775-99)
was purchased from North Coast Medical (Morgan Hill, California).
American Type Culture Collection (Manassas, Virginia) The μ-opioid receptor
rabbit monoclonal antibody (ab134054) was purchased from Abcam (Cambridge,
Massachusetts). The GAPDH [5174] was purchased from Cell Signaling Technology
(CST, Danvers, Massachusetts). The PE10 catheter was purchased from Smiths
Medical (Ashford, Kent, United Kingdom). The Von Frey fiber probe (NC12775-99)
was purchased from North Coast Medical (Morgan Hill, California).
8
Spinal Cord and DRG Immunofluorescence Staining
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Deeply anesthetized rats were perfused transcardially with 0.9% saline followed by 4% paraformaldehyde. The spinal cord and DRGs at L4-L5 segments were removed and postfixed in 4% paraformaldehyde at 4°C for 24 h. After being postfixed, the tissues were transferred into 30% sucrose at 4°C for 3 days for dehydration. The tissues were sectioned at 15 μm thickness for the DH of the spinal cord and 12 μm for the DRG sections. For immunofluorescence staining, sections were blocked in PBS containing 10% goat serum with 0.3% Triton X-100 at room temperature for 2 h and incubated in the primary antibody at 4°C overnight. Sections were then washed in 0.1 M PBS with 0.05% Triton X-100 (pH 7.6) followed by incubating in the secondary antibody at room temperature for 2 h and washing. Sections were mounted on slides and covered with 90% glycerin for observation under a confocal microscope (AR1; Nikon, Tokyo, Japan). The antibodies used included: anti-MOR (1:1000; Abcam, ab134054, Cambridge, United Kingdom), and anti-HDAC2 (1:250; Abcam, ab12169, Cambridge, United Kingdom).
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