The largest database of trusted experimental protocols

5 protocols using everbrite hardset mounting medium

1

RNAScope Multiplex Fluorescent Assay

Check if the same lab product or an alternative is used in the 5 most similar protocols
RNAScope® technology (Advanced Cell Diagnostics Milan, Italy) was used for in situ RNA hybridization according to the manufacturer's protocol. The sections were washed in 1x PBS + 0.1% triton for 3 × 5 min and then placed on glass slides and air dried. The slides were incubated in warmed pre-treatment 3 (Advanced Cell Diagnostics Milan, Italy) for 5 min, kept at a light boil, and then placed in distilled water. The slides were then subjected to RNAscope® Multiplex Fluorescent Assay by first incubating the probe (mmOlig2, mmCasp3, or mm MOG) for 2 h at 40°C and then pre-forming the amplification steps according to instructions. The slides used for Olig2/EdU labeling were then blocked with 5% normal goat serum in 1x PBS +0.1% triton at room temperature for 1 h, and the Click-iT® assay (described above) was then performed followed staining with the nuclear marker DAPI for 5 min. The sections were washed and mounted (Everbrite Hardset mounting medium, Biotium, Hayward, CA).
+ Open protocol
+ Expand
2

Detecting DNA Damage Foci in GFP-ataxin-3 Transfected Cells

Check if the same lab product or an alternative is used in the 5 most similar protocols
GFP-tagged ataxin-3 constructs were transfected in SH-SY5Y neuroblasts grown on collagen-coated 18 mm glass cover slips. 24 Hours after transfection, cells were fixed with 4% paraformaldehyde for 10 minutes and washed with PBS containing 0.5% bovine serum albumin (Sigma-Aldrich) and 0.15% glycine (Thermo Fisher Scientific) for 15 minutes. Slides were then incubated with rabbit anti-53BP1 at 1:1000 (Novus Biologicals, Littleton, USA) for 1.5 hours. After washing, detection was performed by incubation with anti-rabbit IgG Alexa 546 1:1000 (Invitrogen) for 1 hour and mounted with EverBrite hardset mounting medium containing DAPI (Biotium, Hayward, USA). Images of >50 cells were obtained the next day using an AxioImager M2 (Zeiss, Oberkochen, Germany) equipped with a 63x PLAN APO (1.4 NA) oil-immersion objective and an HXP 120 metal-halide lamp used for excitation. Foci were quantified using an ImageJ custom-made macro as described previously47 (link). Only cells expressing the GFP proteins were included for analysis.
+ Open protocol
+ Expand
3

Immunohistochemical Analysis of Sciatic Nerve

Check if the same lab product or an alternative is used in the 5 most similar protocols
The rats were euthanized with isofluorane (3.5–5%, 3.9–5.9 L/min) at the end of the experiment, and the sciatic nerve was harvested and fixed with 10% formalin at room temperature overnight. The sample was embedded in paraffin and pasted on a glass slide (thickness = 7 μm), followed by the removal of paraffin, rehydration, and antigen retrieval (120 °C, 10 min). The sections were washed with tris buffered saline (TBS) that contained 0.025% Triton X-100 for 10 min and were blocked with 10% newborn calf serum and 1% bovine serum albumin in TBS for 2 h at room temperature. Primary antibodies were added to the sections to react overnight at 4 °C followed by the addition of fluorescence-labeled secondary antibodies. After 2 h at room temperature, the samples were washed with TBS and mounted with the EverBrite™ hardset mounting medium that contained DAPI to label the nuclei (1:5000, 40043, Biotium, Fremont, CA). The following primary antibodies were used: anti-MBP (1:1000, ab40390, Abcam, Cambridge, UK) and anti-GFAP (1:1000, Thermo Scientific, Waltham, MA, USA). The secondary antibodies used were Alexa Fluor 555-conjugated goat antimouse IgG (1:100; Thermo Scientific, Waltham, MA, USA) and Alexa Fluor 488-conjugated goat antirabbit IgG (1:100; Thermo Scientific, Waltham, MA, USA). Slides were viewed and images were captured with an LSM780 confocal microscope (Zeiss, Jena, Germany).
+ Open protocol
+ Expand
4

Immunofluorescence Staining of Cultured Cells

Check if the same lab product or an alternative is used in the 5 most similar protocols
Cells (5×103) were seeded into chamber culture slides (BD Falcon, Franklin Lakes, NJ, USA) and fixed with 4% paraformaldehyde in PBS for 10 min at room temperature. After being washed with PBS, the fixed cells were incubated with primary antibodies overnight at 4℃. The cells were then washed three times with cold PBS and cultured with secondary antibody conjugated with goat anti-rabbit IgG-FITC (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at room temperature for 1 h.
Nuclear staining was performed with 4′,6-diamidino-2-phenylindole (DAPI; DAKO, Glostrup, Denmark) for 10 min at room temperature in the dark. The cells were mounted on microscopic slides using EverBrite™ Hardset mounting medium (Biotium, Hayward, CA, USA). Immunolabeling was observed under confocal microscopy (Nikon, Tokyo, Japan).
+ Open protocol
+ Expand
5

Visualizing Nematode Worms via Microscopy

Check if the same lab product or an alternative is used in the 5 most similar protocols
Adult worms were placed on agarose pads with a 10 μL drop of S-basal and covered with a coverslip. The worms were imaged on an Olympus IX83 inverted microscope equipped with a Hamamatsu Orca-Flash 4.0 camera, using a 10× UPLSAPO objective and managed by Olympus CellSens software. For DAPI staining, worms were fixed in ice-cold methanol for 5 min before being mounted in EverBrite™ Hardset Mounting medium (Biotium 23004).
+ Open protocol
+ Expand

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

Sign up now

Revolutionizing how scientists
search and build protocols!