The RNA‐FISH probes targeting RP11‐367G18.1 variant 2 were designed using a Stellaris Probe Designer and were purchased from LGC Biosearch Technologies (Table S4 ). Briefly, cells or tissues were fixed with 4% formaldehyde for 15 min at room temperature, washed, and permeabilized as previously described.18 Samples were incubated in a hybridization solution with RNA‐FISH probes. Hybridization was performed for 4 h at 55°C avoiding light. Samples were washed for three times, blocked with 1% BSA for 1 h, and subsequently incubated with anti‐HIF‐1α (1:100 dilution, abcam) or anti‐H4K16Ac (1:250 dilution, abcam) antibody at 4°C overnight. Then, samples were washed and incubated with secondary antibody solution (1:500 dilution; goat anti‐rabbit Alexa488, abcam) containing a 1:10000 dilution of the DAPI stock solution for 1 h at room temperature. Images were acquired using a Leica TCS SP8 STED microscope and analyzed using MetaMorph software version 7.8.0.0 (Universal Imaging) and Leica Application Suite X (LAS X version 3.5.5.19976) software. RP11‐367G18.1 variant 2 in cells were counted from three independent replicates.
Metamorph software version 7
Manufactured by Universal Imaging
Metamorph software version 7.8.0.0 is an image analysis and processing software. It provides tools for managing, visualizing, and analyzing digital images.
Automatically generated - may contain errors
Lab products found in correlation
Leica tcs sp8 sted microscope, by Leica camera (1 mentions)
Goat anti rabbit alexa 488, by Abcam (1 mentions)
Anti h4k16ac, by Abcam (1 mentions)
Stellaris probe designer, by Biosearch Technologies (1 mentions)
Leica application suite x, by Leica camera (1 mentions)
Anti hif 1α, by Abcam (1 mentions)
C2 fluorescence microscope, by Nikon (1 mentions)
C2 laser scanning confocal microscope, by Nikon (1 mentions)
3 protocols using metamorph software version 7
1
RNA-FISH Assay for RP11-367G18.1 Variant 2
2
Quantitative Analysis of Fluorescent Worm Endosomes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Live worms were mounted onto 2% agarose pads with 100 mM levamisole. Monofluorescence and multiwavelength fluorescence images were acquired at 20°C using a C2 laser scanning confocal microscope equipped with a 100× 1.2-NA oil-immersion objective and NIS-Elements AR 4.40.00 software. Z-series of optical sections were acquired using a 0.5 µm step size.
Monofluorescence images were analyzed by Metamorph software version 7.8.0.0 (Universal Imaging). The “Integrated Morphometry Analysis” function of Metamorph software was employed to measure the fluorescent intensity that is significantly brighter than the background (total intensity), fluorescence area (total area), and puncta number (structure count) within unit regions. From a total of six animals of each genotype, “total intensity,” “structure count,” and “total area” were sampled in three randomly selected unit regions of each animal defined by a 100 × 100 (pixel2) box positioned at random (n = 18 each). In the current study, total area was used to compare tubularity, as the endosomal tubule covers more area than when the tubular network collapses into puncta. Quantification of colocalization images was performed using the open source Fiji (ImageJ) software (Schindelin et al., 2012 (link)). Pearson’s correlation coefficients for GFP and mCherry signals were calculated, and six animals for each genotype were assessed.
Monofluorescence images were analyzed by Metamorph software version 7.8.0.0 (Universal Imaging). The “Integrated Morphometry Analysis” function of Metamorph software was employed to measure the fluorescent intensity that is significantly brighter than the background (total intensity), fluorescence area (total area), and puncta number (structure count) within unit regions. From a total of six animals of each genotype, “total intensity,” “structure count,” and “total area” were sampled in three randomly selected unit regions of each animal defined by a 100 × 100 (pixel2) box positioned at random (n = 18 each). In the current study, total area was used to compare tubularity, as the endosomal tubule covers more area than when the tubular network collapses into puncta. Quantification of colocalization images was performed using the open source Fiji (ImageJ) software (Schindelin et al., 2012 (link)). Pearson’s correlation coefficients for GFP and mCherry signals were calculated, and six animals for each genotype were assessed.
3
Confocal Imaging of Intestinal Autofluorescence
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For confocal fluorescence imaging, young adult animals were mounted on 2% agarose pads with 100 mM levamisole and imaged using C2 + confocal laser scanning microscope (Nikon, Tokyo, Japan) equipped with a 100 3 N.A. 1.45 oil-immersion objective. Z series of optical sections were acquired using a 0.5 mm step size. Multi-wavelength fluorescence images were obtained using a C2 + fluorescence microscope (Nikon, Tokyo, Japan) equipped with a 60 3 N.A. 1.4 oil-immersion objective. Images were captured using NIS-Elements Ver.4.4 software. DAPI channel images were taken to identify broad-spectrum intestinal autofluorescence (Shi et al., 2012) . Quantification of images was performed with Metamorph software version 7.8.0.0 (Universal Imaging, West Chester, PA). GFP/RFP colocalization experiments were performed on L3 and L4 larvae expressing GFP and RFP markers.
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!