Sections were treated with 10 mM citrate (pH 6) of 65 °C at room temperature (RT) for 30 min. After three washes with PBS (5 min each), 10 mM glycine (in PBS) was applied for 20 min, followed by two washes with PBS (5 min). Sections were then permeabilized with 0.3% Triton X-100 for 30 min, washed twice with PBS (5 min) and pre-incubated in blocking buffer (SuperBlock®, ThermoFisher Scientific Prod# 37,515) for 30 min. Subsequently, they were incubated under gentle agitation at 4 °C overnight with each of the following primary antibodies: mouse monoclonal anti-KOR (1:25, 1:50) (ab201552, abcam), anti-Tyrosine hydroxylase Alexa Fluor 488 (1:100) (MAB318-af488, Millipore), rabbit anti-D4R (orb39453, Biorbyt), rabbit anti-D1R (orb107494, Biorbyt), mouse anti-D2R (1:600) (MABN53, Millipore) and rabbit monoclonal anti-SSTR2 (1:100) (ab134252, abcam). After two washes (5 min) with 1:2 dilution of blocking buffer in PBS, slices were incubated for 2 h at 37 °C with the secondary antibodies Alexa Fluor 488-conjugated goat anti-mouse (A11001) or anti-rabbit (A11034, A11008) (1:200, Invitrogen). Following two washes with the diluted blocking buffer in the dark, sections were mounted on SuperFrost glass slides with the addition of mounting medium (Duolink® In Situ Mounting Medium with DAPI, DUO82040, Sigma-Aldrich) and stored at − 20 °C.
Duo82040
Manufactured by Merck Group
Sourced in United States
The DUO82040 is a laboratory equipment product manufactured by Merck Group. It is designed for general laboratory use, but a detailed unbiased description of its core function is not available.
Automatically generated - may contain errors
Lab products found in correlation
Duo92002, by Merck Group (2 mentions)
Duo92004, by Merck Group (2 mentions)
Duo92008, by Merck Group (2 mentions)
Duo82049, by Merck Group (2 mentions)
Lsm 880 confocal microscope, by Zeiss (2 mentions)
Lsm 700 confocal microscope, by Zeiss (2 mentions)
A11034, by Thermo Fisher Scientific (1 mentions)
Mabn53, by Merck Group (1 mentions)
Mab318 af488, by Merck Group (1 mentions)
A11008, by Thermo Fisher Scientific (1 mentions)
Alexa 488 green, by Cell Signaling Technology (1 mentions)
Axioscope 5 microscope, by Zeiss (1 mentions)
Dako buffer, by Agilent Technologies (1 mentions)
Alexa 555 red, by Cell Signaling Technology (1 mentions)
Lsm 780 nlo confocal microscope, by Zeiss (1 mentions)
Ab86146, by Abcam (1 mentions)
Hpa020352, by Merck Group (1 mentions)
Sc 137214, by Santa Cruz Biotechnology (1 mentions)
F3165, by Merck Group (1 mentions)
Duo92101, by Merck Group (1 mentions)
17 protocols using duo82040
1
Immunohistochemical Analysis of Neuroreceptors
2
LCO Staining of Brain Cryosections
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The synthesis of LCOs (qFTAA and hFTAA) was previously described (27 (link)). Brain cryosections (12 μm thick) derived from the brain cortex were thawed at room temperature (RT) for 1 h, fixed in 100% ethanol for 10 min, followed by 70% ethanol for 5 min, subsequently, treated with double-distilled water for 5 min. Next, they were immersed in PBS for 15 min, stained by adding either 1.5 μM hFTAA or qFTAA in PBS dropwise to tissue sections for 30 min at RT, and then rinsed by PBS for 3 × 5 min. They were then allowed to dry under ambient conditions, mounted with the DAKO mounting medium (Sigma–Aldrich; DUO82040), sealed with a nail polish, and settled overnight for further experimental purposes (31 ).
3
FITC-labeled STR Uptake in HeLa Cells
4
Immunofluorescence and Immunohistochemistry Protocols
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For IF staining, cells were grown on glass coverslips, then fixed with methanol for 5 min at room temperature. Following fixation, cells were blocked with Dako buffer (S0809, Agilent) for 1 h, and incubated with primary antibodies overnight at 4 °C, then with appropriate secondary antibodies conjugated with Alexa 555 (red) or Alexa 488 (green) (Cell Signaling Technology). Cells were counterstained with DAPI (DUO82040, Sigma-Aldrich) for 10 min and visualized by fluorescence microscopy (Eclipse-NiE NIKON microscope).
Xenografts were collected and fixed in 4% PFA prior to paraffin embedding, sectioning, staining with hematoxylin and eosin, and with immunostaining conducted as described previously [23 (link)]. Images were acquired on a ZEISS Axioscope 5 microscope.
Xenografts were collected and fixed in 4% PFA prior to paraffin embedding, sectioning, staining with hematoxylin and eosin, and with immunostaining conducted as described previously [23 (link)]. Images were acquired on a ZEISS Axioscope 5 microscope.
5
Proximity Ligation Assay on HepG2 Cells
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PLA was performed on infected HepG2hNTCP cells according to the manufacturer’s instructions (DUO92014, DUO92004, DUO92002, and DUO82040; Duolink; Sigma-Aldrich, St Louis, MO). Briefly, after fixation by 4% paraformaldehyde for 30 minutes at room temperature, and quenching by 1 mol/L glycine, cells were permeabilized for 30 minutes at room temperature with PBS-Triton 0.3%, and then blocked for 30 minutes at 37°C with blocking buffer. Cells were incubated with the 2 primary antibodies overnight at 4°C (Table 2 ). PLA probes were diluted and added to the coverslips for 1 hour at 37°C. After a ligation step of 30 minutes at 37°C, the amplification was performed during 100 minutes at 37°C. Finally, coverslips were mounted onto slips with Duolink In Situ Mounting Medium with 4′,6-diamidino-2-phenylindole (DUO82040-5ML; Sigma). Images were acquired with the Zeiss LSM 780 NLO confocal microscope.
6
Proximity Ligation Assay for Protein Interactions
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HepG2 cells were seeded on an 8-well chamber slide (154534, Thermo Fisher Scientific). After three washes with PBS, the cells were fixed with 4% paraformaldehyde for 15 min at room temperature. Then, the cells were incubated with 1 × Permeabilization Buffer (00-8333-56, Thermo Fisher Scientific) for 15 min, followed by blocked with Duolink block solution for 1 h at room temperature and incubated overnight at 4 °C with the following antibodies: (1) mouse anti-Flag (F3165, Sigma-Aldrich) and rabbit anti-eIF3a (ab86146, Abcam); (2) rabbit anti-METTL16 (HPA020352, Sigma-Aldrich) and mouse anti-eIF3b (sc-137214, Santa Cruz Biotechnology). The next day, cells were washed twice with a large volume of PBS and incubated in PLA probes (DUO92002 and DUO92004, Sigma-Aldrich) for 1 h at 37 °C. Then, the cells were washed with 1 × Duolink for two times In Situ Wash Buffer A (DUO82049, Sigma-Aldrich) and incubated with ligation mix at 37 °C for 30 min. Subsequently, the cells were washed with 1 × Duolink In situ Wash Buffer A twice and incubated with amplification mix (DUO92008, Sigma-Aldrich) at 37 °C for 100 min. Finally, the cells were washed twice with 1 × Duolink in situ wash buffer B, washed once with 0.01 × Buffer B and mounted with Duolink in situ mounting medium with DAPI (DUO82040, Sigma-Aldrich). The pictures were captured under LSM 880 confocal microscope (Zeiss, Germany).
7
Immunofluorescence Imaging of Protein Interactions
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HeLa cells were cultured on coverslips. Following PMA treatment at the specified time and dose, the cells were fixed using 3% paraformaldehyde for 10 min. Subsequently, the cells were permeabilized using 0.1% Triton X-100 in PBS for 5 min, followed by a 30-min blocking step utilizing 2% BSA in PBS. The fixed cells were subjected to a 2-h incubation with the designated primary antibody, after which they were further incubated with an appropriate fluorescence-conjugated secondary antibody for 20 min at room temperature. To mount the cells, mounting media from Sigma (DUO82040) was used. In the case of Duolink® PLA Fluorescence, the cells underwent the same fixation and permeabilization procedure as immunofluorescence, adhering to the manufacturer’s instructions from Sigma (DUO92101). Fluorescence microscopy was conducted using a Zeiss LSM 700 confocal microscope along with ZEN 2012 software (version 1.1.2.0). The acquired images were subsequently processed using Adobe Photoshop.
8
Measuring Src-ERα36 Colocalization in BPA Response
9
Visualizing Protein-Protein Interactions in Cancer Cells
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In situ PLA was performed to visualize protein-protein interactions in MDA-MB-231 and A549 cells. Briefly, cells were seeded onto 3-cm dishes overnight for adherence with ~80% confluence. Cells were then washed with PBS twice, and fixed with 4% paraformaldehyde for 15 min at room temperature. The fixed cells were permeabilized with PBS containing 1% Triton X-100 (Calbiochem, 9410-OP) for 30 min, subsequently blocked in Blocking Solution (Sigma-Aldrich, DUO82007) at 37°C for 1 h and incubated with primary antibodies (anti-BIRC5 [Cell Signaling Technology, 2808]; anti-ATG5 [Millipore, MAB2605; Gene Tex, GTX113309]; anti-ATG12 [Gene Tex, GTX629815], and anti-ATG16L1 [Millipore, ABC25]) overnight at 4°C. On the following day, cells were washed twice with washing buffer (Sigma-Aldrich, DUO82049), and incubated with PLA probes in a ratio of 1:5 in antibody diluent for 1 h at 37°C. The cells were then incubated with ligation solution at 37°C for 30 min and subsequently with amplification solution at 37°C for 100 min. Duolink in situ mounting medium (Sigma-Aldrich, DUO82040) together with DAPI were added to the cells at room temperature for 10 min. Cell images were captured with a confocal microscope (FV1000, Olympus).
10
Protein-Protein Interaction Mapping in HDLECs
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Subconfluent HDLECs were fixed in 4% (v/v) paraformaldehyde in PBS, permeabilized in 0.3% Triton and blocked in 2% (w/v) BSA/PBS 0.05% Tween for 1 h. Primary antibodies [TRBP (Abcam, ab42018) 2.5 μg/ml, S6K2 (Abnova, B02P), S6K1 (Abcam, ab119252), 2.5 μg/ml, IgG Mouse (Cell Signaling) 12.5 μg/ml, IgG Rabbit (Cell Signaling) 2.5 μg/ml and Dicer (Abcam) 20 μg/ml)] were incubated overnight at 4°C in blocking buffer. Washes were performed in blocking buffer, followed by a final wash in 10% blocking buffer/PBS. PLA was performed using Duolink, as per the manufacturer's instructions (Olink Biosciences, Sigma, DUO92102). Cells were stained for F-actin with Alexafluor 488 Phalloidin (Life Technologies, A12379) 1:40 (v/v) in blocking buffer for 20 min, and then mounted in DAPI-containing mounting media, for nuclear staining (Sigma, DUO82040). Images were taken using a Zeiss LSM 510 inverted confocal microscope and the number of PLA events per cell in 100 cells per condition was counted.
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