Embryos were fixed in 4% PFA for 16 hr at 4°C, submerged in 10, 20, and 30% sucrose, embedded in OCT, and sectioned coronally on a cryostat at 12 µm. RNAscope in situ hybridization was performed with RNA probes for the mouse Wnt3a gene (NM_009522.2 , 20 ZZ pairs, target location: 667–1634) and the mouse Ccdc3 gene (NM_028804.1, 9 ZZ pairs, target location: 2–702) and the RNAscope 2.5 HD Reagent Kit-RED (Advanced Cell Diagnostics, USA). Slides were boiled in 1× Target retrieval solution for 5 min, treated with protease for 15 min at 40°C, and incubated with the RNA probe for 2 hr at 40°C. The unbound probe was removed by rinsing slides in 1× wash buffer, and signal amplification reagents were sequentially added. Finally, slides were washed twice in PBS, mounted in Fluoro-Gel medium, and cover slips were applied (Electron Microscopy Sciences, USA). When slides from in utero electroporated embryos were used, GFP fluorescence in each section was imaged to identify electroporated cells prior to the beginning of in situ hybridization.
Fluoro gel medium
Manufactured by Electron Microscopy Sciences
Sourced in United States
Fluoro-Gel medium is a mounting medium designed for the preservation and visualization of fluorescently-labeled samples. It is a water-based, non-hardening gel that helps maintain the fluorescent signal of the sample while providing a protective environment.
Automatically generated - may contain errors
Lab products found in correlation
Superfrost slide, by Thermo Fisher Scientific (1 mentions)
Bx51 microscope, by Olympus (1 mentions)
Mglur5, by Merck Group (1 mentions)
Axiovert m200 inverted microscope, by Zeiss (1 mentions)
Axiocam hr, by Zeiss (1 mentions)
Neutral buffered formalin solution, by Merck Group (1 mentions)
Bx40 microscope, by Olympus (1 mentions)
A4099, by ITW Reagents (1 mentions)
5 protocols using fluoro gel medium
1
Wnt3a and Ccdc3 Expression Analysis in Mouse Embryos
2
Spinal Cord Injury Immunohistochemistry Assay
3
In vivo caspase-3 activity assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mice were anesthetized and via i.n. administrated
with 50 μL of NucView 488 caspase-3 substrate (Ozyme) diluted
in PBS (250×). One hour after injection, mice were euthanized
to harvest lungs for histological analysis. In detail, lungs were
fixed overnight at 4 °C with 10% neutral buffered formalin solution
(Sigma-Aldrich), washed in PBS, and incubated overnight at RT in a
20% PBS/sucrose solution under vacuum. Tissues were then embedded
in the Tissue-Tek OCT compound (Sakura) and frozen in liquid nitrogen,
and cryostat sections (10 μm) were prepared. For staining, tissue
sections were rehydrated in PBS and incubated in a PBS solution containing
1% blocking reagent (Boeringer) (PBS-BR 1%) and DAPI nucleic acid
stain for 20 min. Slides were mounted in Fluoro-Gel medium (Electron
Microscopy Sciences, Hatfield, PA, USA). Labeled tissue sections were
visualized with an Axiovert M200 inverted microscope (Zeiss, Iena,
Germany) equipped with a high-resolution monochrome camera (AxioCam
HR, Zeiss). At least three slides were analyzed per organ from three
different animals, and the results are representative of two independent
experiments.
with 50 μL of NucView 488 caspase-3 substrate (Ozyme) diluted
in PBS (250×). One hour after injection, mice were euthanized
to harvest lungs for histological analysis. In detail, lungs were
fixed overnight at 4 °C with 10% neutral buffered formalin solution
(Sigma-Aldrich), washed in PBS, and incubated overnight at RT in a
20% PBS/sucrose solution under vacuum. Tissues were then embedded
in the Tissue-Tek OCT compound (Sakura) and frozen in liquid nitrogen,
and cryostat sections (10 μm) were prepared. For staining, tissue
sections were rehydrated in PBS and incubated in a PBS solution containing
1% blocking reagent (Boeringer) (PBS-BR 1%) and DAPI nucleic acid
stain for 20 min. Slides were mounted in Fluoro-Gel medium (Electron
Microscopy Sciences, Hatfield, PA, USA). Labeled tissue sections were
visualized with an Axiovert M200 inverted microscope (Zeiss, Iena,
Germany) equipped with a high-resolution monochrome camera (AxioCam
HR, Zeiss). At least three slides were analyzed per organ from three
different animals, and the results are representative of two independent
experiments.
4
Osteoclast Quantification via TRAP Staining
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The TRAP staining protocol was adapted from a previous report.55 (link) EDTA-decalcified tissue cryosections were air-dried at room temperature for 30 min. The slides were rehydrated by three 5-min rinses with PBS and incubated with the TRAP staining solution for 30 min or until the positive colour developed in a 37 °C water bath, according to the manufacturer’s protocol (Sigma-Aldrich, 387A). The slides were counterstained with Fast Green solution for 20 min and mounted with Fluoro-gel medium (Electron Microscopy Sciences, 50–247–04). Multinucleated TRAP-positive osteoclasts (three or more nuclei) were counted using five slides per mouse under a light microscope. Double-labelling analysis of LacZ and TRAP was performed by TRAP staining of the X-gal-stained sections.
5
Cell Attachment Quantification Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells (1 x 10 4 cells/coverslip) were plated on 13 mm round glass coverslips and incubated for 1 hour at 37 ºC in 5% CO 2 prior to fixation, each sample was studied in triplicate. The cells were then washed with PBS and fixed for 20 min with 4% PFA (Sigma, USA) in PBS. The nuclei were stained with DAPI (1:5,000 dilution, Panreac Applichem A4099, Spain) and all the coverslips were mounted with Fluoro-Gel medium (Electron Microscopy Sciences, USA). The images were acquired on an Olympus BX40 microscope equipped with an Olympus DP73 camera (Spain) at 20X and the total number of cells attached to the coverslips was quantified.
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!