The FISH procedure was performed according to previously published methods [47 (link)]. 7 μm cryo-sections were cut for hybridizations. Lgr5 mRNA Stellaris probe (mouse: NM_010195) was designed by Probe Designer at www.singlemoleculefish.com and synthesized by Biosearch Technologies. The FISH probe set consists of 96 TMR fluorophore labelled oligonucleotides. DAPI nuclear dye was included during the final wash. Images were captured at 405nm and 568nm using 100x objective len by an Olympus FV1000 upright confocal microscope.
Fv1000 upright confocal microscope
Manufactured by Olympus
Sourced in Japan, United Kingdom
The FV1000 is an upright confocal microscope designed for high-resolution imaging of biological samples. It features a modular design and supports multiple laser lines for fluorescence excitation. The FV1000 enables optical sectioning and three-dimensional reconstruction of samples.
Automatically generated - may contain errors
Lab products found in correlation
Prolong diamond antifade mountant with dapi, by Thermo Fisher Scientific (2 mentions)
Fv3000 fluoview inverted confocal microscope, by Olympus (2 mentions)
Fv31s swpad software, by Olympus (2 mentions)
Fv10 asw software, by Olympus (2 mentions)
Axio imager upright microscope, by Zeiss (1 mentions)
Sc 7907, by Santa Cruz Biotechnology (1 mentions)
Bond max, by Leica (1 mentions)
Low melting point agarose, by Merck Group (1 mentions)
Millicell ez 4 well glass slides, by Merck Group (1 mentions)
Prolong diamond antifade mountant, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 dye, by Thermo Fisher Scientific (1 mentions)
Poly l lysine (pll), by Merck Group (1 mentions)
Alexa fluor dye, by Thermo Fisher Scientific (1 mentions)
4 well chamber slide, by Merck Group (1 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (1 mentions)
S2369, by Agilent Technologies (1 mentions)
S2367, by Agilent Technologies (1 mentions)
Envisionc, by Agilent Technologies (1 mentions)
Cy3 conjugated anti rat igg, by Thermo Fisher Scientific (1 mentions)
16 protocols using fv1000 upright confocal microscope
1
Single Molecule FISH of Lgr5 mRNA
2
Immunohistochemical Staining for p53
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immunostaining was performed on formalin-fixed paraffin-embedded (FFPE) 5 μm sections. We used commercial rabbit anti-p53 (CM5, 1:500, Leica Biosystems, Germany) primary antibody and the p53 IHC procedure was performed by auto-staining machine (Leica Bond-max, Leica Biosystems, Germany) to ensure reproducibility of staining between experiments, the procedure was performed according to the manufacturer's instructions. The most critical step for p53 IHC staining is that we used EDTA based pH 9.0 antigen retrieval solution to exposure antigen epitopes. Home-made polyclonal rabbit anti-p53, commercial monoclonal mouse anti-p53 (1C12, 1:200, #2425S, Cell Signaling Technology, Danvers, USA), rabbit anti-PCNA (1:100, sc-7907, Santa Cruz), and mouse anti-p21 (1:20, F5, Santa Cruz) primary antibodies and anti-rabbit/mouse Alexa Fluor 568/488 (1:500, Invitrogen, California, USA) secondary antibodies were used for immunofluorescent staining. IHC Images were captured with a Zeiss AxioImager upright microscope using 20x and 40x objective lens. Data presented are representative of results obtained from at least 3 mice per group. Immunofluorescent staining images were observed using an Olympus FV1000 upright confocal microscope and captured at 405nm, 488nm, and 568nm using 40x and 100x objective lens, processing with FV10-ASW 3.0 Viewer software.
3
Comparative Image Analysis of Spheroids
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Image analysis using ImageJ was carried out on the data sets acquired on the Olympus FV1000 upright confocal microscope to compare the performance of the various immersion media. For each z-stack from representative day 5 spheroids immersed in glycerol, ScaleView-A2 or PBS, a region of interest that represented the area of the whole spheroid was created. A specialised plugin for ImageJ developed by Maske18 was used to derive the raw integrated density, which describes the strength of the signal and is determined by the sum of the values of the pixel within the specific area. The maximum depth of imaging (or penetration depth) was determined as the point where no fluorescence signal above background was detected, i.e., where there were no pixel values above the measured mean background value.
4
Imaging Pyloric Epithelial Tissue Morphology
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Samples of the lesser and greater part of the pyloric epithelium were identified anatomically as described in Supplementary Figure 7 .
“xy plane” side-view imaging was performed using semithick sections of near-native tissue, generated as described in3 (link).
“xz plane” bottom-view imaging was performed after removal of the muscle layer, and the epithelial tissue was directly mounted upside down in Hydromount containing Hoechst dye nuclear counterstain.
Images were acquired using an Olympus FV1000 upright confocal microscope.
“xy plane” side-view imaging was performed using semithick sections of near-native tissue, generated as described in3 (link).
“xz plane” bottom-view imaging was performed after removal of the muscle layer, and the epithelial tissue was directly mounted upside down in Hydromount containing Hoechst dye nuclear counterstain.
Images were acquired using an Olympus FV1000 upright confocal microscope.
5
Co-localization of ABIN1 and Tat in HeLa Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For co-localization staining of ABIN1 and Tat, HeLa cells cultured on slides were transfected with ABIN1 and Tat expressing constructs for 24 h, then washed three times with PBS, fixed for 20 min in 4% paraformaldehyde at room temperature, followed by twice wash with ice-cold PBS and permeating with 0.2% TritonX-100 in PBS for 30 min, then blocked with blocking buffer (3% BSA in PBS) for 30 min at room temperature after another twice wash. Primary antibodies were diluted with 1% BSA in PBS and incubated with cells at room temperature for 2 h. After three times wash with PBS to remove the non-associated antibodies, cells were incubated with secondary antibodies conjugated with FITC (Fluorescein) (Thermo Scientific) and Rhodamine (Thermo Scientific) for 1 h at room temperature. Cells were then washed for three times with PBS and slides were mounted with Prolong Diamond Antifade Mountant with DAPI (Thermo Scientific) and viewed on Olympus FV1000 upright confocal microscope, using FV10-ASW 1.7 software to acquire images.
6
Live Imaging of Zebrafish Larvae
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For live imaging, 5-dpf larvae were embedded in 1.5% low melting-point agarose (Sigma) at 28.5°C automatic thermostats. Larvae were placed in a dorsal view without anesthetics. Images were carried out under a Zeiss 40 × NA 0.80 water immersion objective with an Olympus FV1000 upright confocal microscope (473 nm, 543 nm; Japan). The image stacks were in the range of 40–65 μm in depth, captured at 1 μm/optical section at a 5-min interval.
7
Immunofluorescence Staining of HEK293T Cells
8
Immunofluorescence Staining of NPC Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
NPCs were placed in 4-well chamber slides (Merck Millipore) coated with Matrigel. Cells were fixed in 4% paraformaldehyde for 30 min at room temperature. After three washes in PBS, cells were permeabilized in 0.5% Triton X-100 for 15 min at room temperature. After three washes in PBS, cells were blocked in 1% BSA for 30 min at room temperature. CO sections were placed in an oven at 60°C for 45 min. Slides were cooled down at room temperature, followed by a deparaffinization procedure in xylene and ethanol. After being washed in water for 5 min, slides were immersed in 10% antigen retriever (citrate buffer, pH 6, Sigma-Aldrich; C9999) and placed in a pressure cooker for 30 min. After slides were cooled down at room temperature (90 min) and washed three times in PBS, they were blocked in blocking buffer (1% BSA and 0.5% Triton X-100). Primary antibodies were prepared in 1% BSA and 0.5% Triton X-100 and added accordingly for overnight incubation at 4°C. After several washes in PBS, secondary antibodies labeled with Alexa Fluor dyes (Life Technologies) were incubated for 1 h at room temperature. After three washes in PBS, cells were stained with DAPI. Slides were mounted in ProLong Gold Antifade Reagent (Life Technologies). Images were taken with bright-field and wide-field Zeiss AxioImager Z1 upright microscopes or an Olympus FV1000 upright confocal microscope.
9
Collagen Coating and Cell Adhesion Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Collagen (Roche) at 20 µg/ml was coated onto sterile coverslips at 37ºC for 1 hr in 12 well plates. After washing with PBS, coverslips were blocked with 0.5% BSA in PBS for 20 min. Cells (1.5x104) were seeded onto each coverslips for 20 hr. The cells were then fixed with 4% paraformaldehyde/cytoskeletal buffer (100 mM KCl, 300 mM sucrose, 2 mM EGTA, 2 mM MgCl2, 10 mM PIPES, pH 7.4) for 20 min, then washed three times with PBS. Coverslips were incubated with 0.05% Triton X/PBS solution for 5 min and washed with PBS. BSA (3%) in PBS was used to block the coverslips for 1 hr. Phalloidin was diluted with 3% BSA and incubated with the cells at 4ºC overnight. After three washings with PBS, coverslips were mounted with ProLong Diamond Antifade Mountant with DAPI (P36962, Thermo Fisher Scientific) and images were acquired with Olympus FV1000 upright confocal microscope. To quantify cell morphology (cell area and aspect ratio), individual cells were analyzed using ImageJ software. A minimum of 60 images were analyzed for each cell-line
10
Immunohistochemistry and Immunofluorescence of Mouse Testes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mice testes for IHC and immunofluorescence staining were proceeded as described previously.33 (link), 34 (link) The experiments were repeated three times. The antibodies used for IHC/immunofluorescence staining are listed in Supplementary Information . Specifically, antigen retrieval was carried out by heating slides in a pressure cooker (121 °C) for 10 min in a citrate buffer pH9.0 (S2367, DAKO, Glostrup, Denmark) for p53 staining and pH6.0 (S2369, DAKO) for other antibody staining. The peroxidase-conjugated secondary antibodies used were mouse/rabbit EnVisionC (DAKO) for HRP-immunostaining or anti-rabbit/mouse Alexa 488/568 IgG (Invitrogen, Carlsbad, CA, USA) for immunofluorescence staining. Images were captured with a Zeiss AxioImager upright microscope and Olympus FV1000 upright confocal microscope.34 (link)
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!