The immunohistochemical analyses were performed as described in ref. 61 (link). In brief, brain sections were rinsed 5 times in PBS (Sigma-Aldrich P5368-10pak) for 5 min, and then blocked in 10% normal donkey serum and 0.3% Triton X-100 in PBS to minimize nonspecific binding. The sections were then incubated with the primary antibodies (Supplementary Table 4 ) in the blocking solution for 48 h at 4 °C. The sections were then rinsed in PBS and incubated with a corresponding secondary antibody for 4 h. After the last rinse, the sections were mounted, air dried overnight; and were then sealed with a cover slip in Dako Fluorescence Mounting Medium (S3023, Dako North America, Inc., Carpinteria, CA) and stored at −20 °C.
Dako fluorescence mounting medium
Manufactured by Agilent Technologies
Sourced in United States, Denmark, Germany, Canada, Australia, France, Italy
Dako Fluorescence Mounting Medium is a product designed for use in fluorescence microscopy. It is a mounting medium that helps preserve and protect fluorescent samples during microscopic examination.
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Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (20 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (14 mentions)
Triton x 100, by Merck Group (11 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (10 mentions)
Hoechst 33342, by Thermo Fisher Scientific (10 mentions)
Lsm 880 confocal microscope, by Zeiss (8 mentions)
Hoechst 33342, by Merck Group (4 mentions)
Alexa fluor 647, by Thermo Fisher Scientific (4 mentions)
Bovine serum albumin (bsa), by Merck Group (4 mentions)
Lsm 710 confocal microscope, by Zeiss (4 mentions)
Anti guinea pig alexafluor 647, by Thermo Fisher Scientific (3 mentions)
Cd31 pe, by BioLegend (3 mentions)
Alexa fluor 594, by Thermo Fisher Scientific (3 mentions)
Goat serum, by Thermo Fisher Scientific (3 mentions)
Bx53 fluorescence microscope, by Olympus (3 mentions)
Alexa fluor 555, by Thermo Fisher Scientific (3 mentions)
Sp5 confocal microscope, by Leica (3 mentions)
Normal goat serum (ngs), by Thermo Fisher Scientific (3 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (3 mentions)
Eclipse c1 plus, by Nikon (3 mentions)
210 protocols using dako fluorescence mounting medium
1
Immunohistochemical Analysis of Brain Sections
2
Immunofluorescence Staining of BEAS-2B Cells
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BEAS-2B cells were seeded on coverslips and incubated overnight in DMEM plus 10% FBS at 37°C with 5% CO2 in a humidified cell incubator. Cells were fixed using 4% paraformaldehyde and permeabilized with 0.1% Triton X-100 in phosphate-buffered saline (PBS). Cells were incubated with anti-XB13 (mouse IgG, homemade) and anti–Tks5 SH3#1 (rabbit IgG; EMD Millipore), primary antibody and secondary antibody conjugated with Alexa Fluor 594 (Molecular Probes, Eugene, OR), or Oregon green 488 (Molecular Probes). Actin was stained using CytoPainter phalloidin-iFluor 405 reagent (Abcam). Coverslips were mounted onto glass slides with Dako fluorescence mounting medium (Dako, Mississauga, Canada). Images were obtained using an Olympus FluoView Confocal FV1000-ASW and analyzed by Olympus FluoView FV10-ASW (Olympus, Tokyo, Japan) and ImageJ (National Institutes of Health, Bethesda, MD).
3
Immunohistochemical Analysis of Enteric Neurons
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Tissue was fixed in 4% formaldehyde overnight at 4 °C, then rinsed for 3 × 10 min in PBS. For colon tissue from mGlu5-KO mice, the mucosa, submucosal plexus, and circular muscle were carefully peeled off, leaving the LMMP. All tissue was permeabilized in 10% CASBLOCK + 0.1% Triton for 30 min and incubated in primary antisera overnight at 4 °C. Tissue was then rinsed in PBS 3 × 10 min, incubated in secondary antisera for 2 h at room temperature, then rinsed again 3× 10 min in PBS prior to being mounted on glass slides in Dako fluorescence mounting medium (Dako, Carpentaria, CA, USA) and secured with a glass coverslip.
Primary antisera used included Human anti-HuC/D (Hu; 1:5000, gift from Dr V. Lennon), Sheep anti-nNOS (1:2000, gift from P. Emson), Rabbit anti-Calbindin (1:1600, Swant #CB-38a), Goat anti-Calretinin (1:1000, Swant #CG1), and Rabbit anti-S100β (1:1000, Dako #Z0311). Secondary antisera included donkey anti-Human Alexa Fluor® 647 (1:500, Jackson #709-605-149), donkey anti-Sheep Alexa Fluor® 488 (1:400, Molecular Probes #A11015) and Alexa Fluor® 647 (1:500, Molecular Probes #A21448), and donkey anti-Rabbit Alexa Fluor® 594 (1:400, Molecular Probes #A21207) and Alexa Fluor® 647 (1:400, Molecular Probes #A31573).
Primary antisera used included Human anti-HuC/D (Hu; 1:5000, gift from Dr V. Lennon), Sheep anti-nNOS (1:2000, gift from P. Emson), Rabbit anti-Calbindin (1:1600, Swant #CB-38a), Goat anti-Calretinin (1:1000, Swant #CG1), and Rabbit anti-S100β (1:1000, Dako #Z0311). Secondary antisera included donkey anti-Human Alexa Fluor® 647 (1:500, Jackson #709-605-149), donkey anti-Sheep Alexa Fluor® 488 (1:400, Molecular Probes #A11015) and Alexa Fluor® 647 (1:500, Molecular Probes #A21448), and donkey anti-Rabbit Alexa Fluor® 594 (1:400, Molecular Probes #A21207) and Alexa Fluor® 647 (1:400, Molecular Probes #A31573).
4
Immunofluorescence Staining of Tissue Sections
5
Immunofluorescent Staining and Imaging of Protein Localization
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Cells were fixed with 4% paraformaldehyde (PFA) and then washed with PBS. For 4°C experiments, cells were maintained at 4°C for 10 minutes before treatments, and maintained at that temperature for an additional 30 minutes before fixation. Nonspecific binding was prevented using Sea Block blocking solution, for 1 hour at RT (Thermo Fisher Scientific). Cells were kept overnight at 4°C in a primary antibody solution and incubated for 1 hour at RT with the corresponding secondary antibody. Antibodies were used as listed: rabbit anti‐Ago‐2 (1:100) (Cell Signaling Technology Inc); rabbit anti‐CD8 (1:50) (Santa Cruz Biotechnology Inc); Alexa Fluor 594 goat anti‐rabbit (1:200) (Molecular Probes, OR). For nuclear labeling, cell preparations were stained with Hoechst‐33342 (2 μg/mL) (Sigma) and mounted in Dako Fluorescence Mounting Medium (Dako North America Inc). Fluorescent images were acquired using an LSM 510 confocal microscope with a 40× objective (Carl Zeiss Inc).
6
Immunofluorescent Localization of TSEN54 in Cerebrum
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Immunofluorescence was performed to visualize the distribution and localization of TSEN54 in the cerebrum according to an established protocol [39 (link)]. For this purpose, a monoclonal mouse anti-human-TSEN54-antibody (anti-TSEN54, sc-398327, dilution 1:50, Santa Cruz Biotechnology, Inc) was used. Following the same procedure as described above, after dewaxing and antigen retrieval, non-specific binding was blocked with 20% goat serum diluted in diluted in PBS/0.1% Triton X/1% BSA. Thereafter the sections were incubated with the primary anti-TSEN54 antibody, diluted in PBS/0.1% Triton X/1% BSA. Sections for negative control were incubated with non-immune serum.
Goat-anti-mouse IgG CyTM3 (115-165-166, dilution 1:200, Jackson ImmunoResearch) was used as secondary antibody. Nuclear counter-staining was performed with 0.01% bisbenzimide (Sigma-Aldrich Chemie GmbH) and sections were mounted with Dako fluorescence mounting medium (DakoCytomation GmbH).
Goat-anti-mouse IgG CyTM3 (115-165-166, dilution 1:200, Jackson ImmunoResearch) was used as secondary antibody. Nuclear counter-staining was performed with 0.01% bisbenzimide (Sigma-Aldrich Chemie GmbH) and sections were mounted with Dako fluorescence mounting medium (DakoCytomation GmbH).
7
In Vivo Peptide Tissue Visualization
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The tissue, including skin and muscle, under the anode was collected after ItP of the FITC-labeled peptide and was embedded in OCT compound (Sakura Finetek Japan Co.,Ltd., Tokyo, Japan), followed by freezing with a dry ice–ethanol bath. Then, frozen tissue sections (10 μm) were prepared with a cryostat. After mounting the sections with Dako Fluorescence Mounting Medium (Dako North America, Inc., Carpinteria, CA, USA), FITC fluorescence in the tissue section was observed by confocal laser scanning microscopy (LSM700, Carl Zeiss, Jena, Germany).
8
Assessing Cell Proliferation via Ki67 Staining
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Cell proliferation levels were assessed using Ki67 staining. HAoEC cells at PD 65 were seeded at 1 x 104 cells /coverslip and after 10 days were treated with Na-GYY4137 (at 100ug/ml) or AP39, AP123, RT01 (at 10ng/ml) for 24 hours in 3 biological replicates. Cells were then fixed for 10 min with 4% v/v PFA and permeabilized with 0.025% v/v Triton and 10% v/v serum in PBS for 1 hour. Cells were incubated with a rabbit monoclonal anti-Ki67 antibody (ab16667, Abcam, UK) at 1:200 overnight at 4°C followed by FITC-conjugated secondary goat anti-rabbit (1:400) for 1 hour, and nuclei were counterstained with DAPI. Coverslips were mounted on slides in DAKO fluorescence mounting medium (S3023; Dako). The proliferation index was determined by counting the percentage of Ki67 positive cells from at least 300 nuclei from each biological replicate at 400× magnification under a Leica D4000 fluorescence microscope. Proliferation was also assessed by cell counts. For this, cells were seeded at 6 x 104 cells per well into 6-well plates and cultured for 10 days then treated with each compound for 24 hours. Cell counts in three replicates of treated and vehicle-only cultures were carried out manually following trypsinisation and suspension of cells and are presented as mean (+/-SD).
9
Quantification of Fluorescence in Microscopy
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Samples were mounted with Dako Fluorescence Mounting Medium (Dako, Carpinteria, CA, USA). Images were acquired by using an FV1200 scanning confocal microscope (Olympus, Tokyo, Japan) with a UPLSAPO40X2 objective.
For quantitation of fluorescence levels, we used single confocal sections through the nucleus. We traced the nucleus manually using ImageJ, then we measured integrated fluorescence intensity. To calculate background levels for the use of a specific primary antibody (anti-mCherry or anti-p27KIP1), we took a similar-sized region of interest, and for each sample measured five unstained tissue regions in the section, then used the average of these regions as the background level. Representative maximum intensity measurements are reported inFigure S2A,B . For the majority of the OE cells in vivo, the 3D organization of the tissue means we are not quantifying from the nucleus at the position of its maximum area. Therefore, we calculated the fluorescence intensity in fluorescence units (FU) as (integrated fluorescent intensity/area) [14 (link)] for comparative measurements.
Statistical analyses were carried out using Prism 6 (GraphPad, San Diego, CA, USA). The Alpha value was p < 0.05. In each experiment, the test and corrections for multiple testing are stated in the figure legend.
For quantitation of fluorescence levels, we used single confocal sections through the nucleus. We traced the nucleus manually using ImageJ, then we measured integrated fluorescence intensity. To calculate background levels for the use of a specific primary antibody (anti-mCherry or anti-p27KIP1), we took a similar-sized region of interest, and for each sample measured five unstained tissue regions in the section, then used the average of these regions as the background level. Representative maximum intensity measurements are reported in
Statistical analyses were carried out using Prism 6 (GraphPad, San Diego, CA, USA). The Alpha value was p < 0.05. In each experiment, the test and corrections for multiple testing are stated in the figure legend.
10
Immunohistochemistry on Lung Tissue
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Five micron-sections of lung tissue or reconstituted ALI epithelium, fixed in 4% formaldehyde and paraffin-embedded, were deparaffinised in toluene and rehydrated through a graded series from ethanol to water. Antigen retrieval was performed in citrate buffer (pH 6• 0 containing 0• 1% of triton) using a pressure cooker at 15 PSI for 5 min. Sections were blocked for non-specific antigen binding by incubation in Bloxall (Vector Laboratories Inc.) for 15 min and then in 0• 3% hydrogen peroxide with 5% goat serum (Bio-Rad) for 30 min. Staining first included a 30 min protein blocking with 5% goat serum, then the primary antibodies diluted in 5% normal goat serum solution were applied, then the appropriate SuperBoost™ goat anti-rabbit or anti-mouse, poly-HRP-conjugated secondary antibody (Thermo Fisher Scientific) was applied for 40 min. HRP-conjugated polymer mediated the focal covalent binding of a fluorophore using tyramide signal amplification. Table S5 recapitulates the antibodies and fluorophores that were used. Finally, sections were counterstained with Hoechst (Thermo Fisher Scientific) diluted at 10 µg/ml in TBS-BSA 5% and mounted with Dako fluorescence mounting medium (Dako, Carpinteria, CA). For negative controls, we used rabbit or mouse isotype controls at the same concentration as the corresponding primary antibodies (diluted in 5% normal goat serum).
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