Aortic tissue segments were transferred into PCR tubes and subjected to RNAscope in a thermocycler with mouse anti-Klf2-C1 (510671; ACD) or mouse anti-Nos3-C1 (443061; ACD) labeled with Cy5 (FP1171; Perkin Elmer) and mouse Cdh5-C2 (132531; ACD) labeled with Cy3 (FP1170; Perkin Elmer) and DAPI. Aortas were then mounted on microscope slides en face. Endothelium, distal to intercostal branchpoints and displaying proper alignment, was imaged by widefield microscopy with a 60× oil objective. To quantify the Klf2-647 signal, background fluorescence was removed in ImageJ using a 1-μm diameter rolling ball radius subtraction followed by despeckling (weighted average of a 3 × 3-pixel grid). The resultant average fluorescence intensity of the field was divided by the number of ECs for three fields/mouse. Six mice were quantified for each group. For in vitro RNAscope labeling, MAEC were left static or subjected to 18 dynes/cm2 laminar shear stress for 16 h before 30 min fixation in 3.7% formaldehyde. Slides were then labeled with Klf2-Cy5 according to the manufacturer’s protocol and imaged using widefield microscopy.
Fp1170
Manufactured by PerkinElmer
The FP1170 is a fluorescence spectrophotometer designed for laboratory applications. It is capable of measuring fluorescence intensity across a range of wavelengths. The device features automated operation and data analysis capabilities.
Automatically generated - may contain errors
Lab products found in correlation
Fp1171, by PerkinElmer (1 mentions)
Digoxigenin labeled probe, by Roche (1 mentions)
Mab3402, by Merck Group (1 mentions)
Fv3000 confocal laser scanning microscope, by Olympus (1 mentions)
Mab1195, by R&D Systems (1 mentions)
Ab62140, by Abcam (1 mentions)
Anti mouse igg alexa 647, by Thermo Fisher Scientific (1 mentions)
Fp1135, by PerkinElmer (1 mentions)
5 protocols using fp1170
1
Visualizing Endothelial Mechanosensors in Aorta
2
In Situ Hybridization of Mouse DRG
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RNA probes were synthesized with gene-specific PCR primers and cDNA templates from mouse DRG. In situ hybridization was performed with digoxigenin-labeled probes (Roche, cat# 11277073910). Probes were incubated with the slides overnight at 55°C and the slides were then incubated with the horseradish peroxidase-conjugated anti-digoxigenin antibody 1:500 (Roche, Cat#11207733910; RRID:AB_514500 ). Final detection was achieved with TSA-Cy3 at a dilution of 1:50 (Perkin Elmer Life Sciences, FP1170). The oligonucleotides used for the nested PCRs and for probe synthesis are listed in the key resources table .
3
Quantification of APOE mRNA and Astrocyte Immunostaining
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Four mice per experimental condition were stained for APOE mRNA by RNAscope. RNAscope experiments were performed using the Manual Fluorescent Multiplex kit v2 (Advanced Cell Diagnostics) following manufacturer’s recommendations with minor adjustments. Briefly for each mouse a single paraffin section was baked onto a superfrost slide for use in APOE mRNA quantification. Following deparaffinization, target retrieval and protease digestion, probe hybridization was carried out at 40°C for 2 h with hs-APOE (433091), 3-plex Positive Control Probe_Mm (320881) and Negative Control Probe- DapB (310043). After amplification steps to obtain the RNAscope signals, the signal was developed using TSA-cy3 (Perkin Elmer FP1170).
We then immediately performed immunohistochemistry. Sections were permeabilized in 0.5% Triton-X for 15 min before being blocked in 0.1% Triton-X and 5% normal goat serum for 1 h at room temperature. Primary antibodies GFAP (Millipore MAB 3402) was diluted 1:1000 in 0.05% and 2.5% normal goat serum overnight at 4°C. Secondary was goat anti-mouse 488 (Thermo-Fisher A11001) diluted 1:500 in 0.05% and 2.5% normal goat serum at room temperature for 1 h. Sections were then mounted using Vectashield Antifade mounting medium with DAPI (ZG0729) cover-slipped and imaged on an Olympus FV3000 confocal laser scanning microscope.
We then immediately performed immunohistochemistry. Sections were permeabilized in 0.5% Triton-X for 15 min before being blocked in 0.1% Triton-X and 5% normal goat serum for 1 h at room temperature. Primary antibodies GFAP (Millipore MAB 3402) was diluted 1:1000 in 0.05% and 2.5% normal goat serum overnight at 4°C. Secondary was goat anti-mouse 488 (Thermo-Fisher A11001) diluted 1:500 in 0.05% and 2.5% normal goat serum at room temperature for 1 h. Sections were then mounted using Vectashield Antifade mounting medium with DAPI (ZG0729) cover-slipped and imaged on an Olympus FV3000 confocal laser scanning microscope.
4
In Situ Hybridization of Rreb1 in Mouse Brain
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We used an RNAScope probe for Rreb1 (ACDBio, 542641-C2) for in situ hybridization on two wild-type and two nm3888−/− mice following the manufacturer’s protocol. Briefly, we transcardially perfused mice with 10% neutral-buffered formalin and processed and embedded brains for paraffin sectioning. We baked 7-μm sections at 60°C for 1 hour before deparaffinization. We treated sections with hydrogen peroxide (ACDBio, 322281) before antigen retrieval in target retrieval buffer (ACDBio, 322000) and treatment with protease plus (ACDBio, 322281). We incubated sections with the probe for 30 min and then washed them in 1× wash buffer (ACDBio, no. 310091). We stored slides in 5× SSC overnight before a series of amplification steps and incubation with Cy3 (PerkinElmer, FP1170, 1:1500). For immunofluorescence after in situ hybridization, we incubated either calbindin D-28 (rabbit, Swant, cb38, 1:1000) or tyrosine hydroxylase (mouse, Leica, NCL-L-TH, 1:250) on slides overnight, then incubated them with secondary antibody. We stained nuclei with DAPI and slides and stored them in the dark at 4°C before imaging.
5
Whole Mount Immunohistochemistry of Lungs
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Lungs were fixed in DMSO:methanol (1:4) at 4 °C with gentle shaking for overnight. Fixed lungs were then incubated in 30 % H 2 O 2 :DMSO:methanol (1:1:4) for 8 hours at room temperature with gentle rocking, and stored at -20 °C in 100 % methanol.
Whole mount immunohistochemistry was performed as previously described (Metzger et al., 2008) with the following modifications: specimens were incubated with anti-VAChT (1:200, Abcam, ab62140) diluted in 0.5 % BR/PBST for 2 or 3 nights at 4 °C, and subsequently with secondary antibodies conjugated with anti-rabbit IgG-Alexa 488, anti-mouse IgG-Alexa 647 (1:500, Molecular Probes), and AmpliStain TM anti-Goat 1-Step HRP (1:500, Stereospecific Detection Technologies, AS-G1-HRP) overnight at 4 °C. The specimens were reacted with 1:200 dilution of Cy3-tyramid (Perkin-Elmer, FP1170) in 1× Amplification diluent (Perkin-Elmer, FP1135) for 1 hour at room temperature, and washed three times in TNT (0.1 M Tris-HCl [pH7.5], 150 mM NaCl, 0.1 % Tween 20) to terminate the reaction. Anti-PGP9.5 (1:200, Invitrogen, 38-1000) and anti-Tuj1 (1:300, R&D systems, MAB1195) were also used for tissues fixed in DMSO:methanol.
Whole mount immunohistochemistry was performed as previously described (Metzger et al., 2008) with the following modifications: specimens were incubated with anti-VAChT (1:200, Abcam, ab62140) diluted in 0.5 % BR/PBST for 2 or 3 nights at 4 °C, and subsequently with secondary antibodies conjugated with anti-rabbit IgG-Alexa 488, anti-mouse IgG-Alexa 647 (1:500, Molecular Probes), and AmpliStain TM anti-Goat 1-Step HRP (1:500, Stereospecific Detection Technologies, AS-G1-HRP) overnight at 4 °C. The specimens were reacted with 1:200 dilution of Cy3-tyramid (Perkin-Elmer, FP1170) in 1× Amplification diluent (Perkin-Elmer, FP1135) for 1 hour at room temperature, and washed three times in TNT (0.1 M Tris-HCl [pH7.5], 150 mM NaCl, 0.1 % Tween 20) to terminate the reaction. Anti-PGP9.5 (1:200, Invitrogen, 38-1000) and anti-Tuj1 (1:300, R&D systems, MAB1195) were also used for tissues fixed in DMSO:methanol.
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