FFPE tibia bones were further sequentially sectioned and baked at 56°C in preparation for immunofluorescence staining of osteoblast (RUNX2 at 1:500; Abcam Cat. No. ab81357) and nuclear staining (DAPI). Deparaffined and rehydrated slides were subject to heat-induced antigen retrieval method. Sections were then blocked and incubated in primary antibodies diluted in 10% normal goat serum in TBS overnight at 4°C. Subsequently, slides were stained with secondary Alexa Fluor 568-conjugated antibody at 1:1000 at room temperature for 1 hour under light-proof conditions. Stained slides were stained with DAPI for nuclear contrast and mounted for imaging at 20X using Zeiss upright fluorescent microscope to include the injury site as well as the immediate peripheral tissue. All RUNX2 positive cells (red staining colocalizing with DAPI) within 5μm radius from injury were counted and mathematically converted to osteoblasts / bone marrow volume (#OBL/μm3) for each bone at each time point.
Upright fluorescent microscope
Manufactured by Zeiss
The Upright Fluorescent Microscope is an optical instrument designed to visualize and analyze fluorescently-labeled samples. It utilizes a vertically-oriented optical path to provide a high-resolution, magnified view of the specimen.
Automatically generated - may contain errors
Lab products found in correlation
Lab tek 2 chamber slide, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions)
Runx2, by Abcam (1 mentions)
Lipofectamine crisprmax cas9 transfection reagent, by Thermo Fisher Scientific (1 mentions)
Normal goat serum (ngs), by Thermo Fisher Scientific (1 mentions)
Alexa conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Hoechst, by Thermo Fisher Scientific (1 mentions)
Click it plus kit, by Thermo Fisher Scientific (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Epcam, by Thermo Fisher Scientific (1 mentions)
Goat anti mouse alexa 488, by Thermo Fisher Scientific (1 mentions)
Prolong gold anti fade reagent containing dapi, by Thermo Fisher Scientific (1 mentions)
Antibody to phospho h2ax, by Merck Group (1 mentions)
Poly l lysine solution, by Merck Group (1 mentions)
11 protocols using upright fluorescent microscope
1
Quantifying Osteoblast Dynamics in Tibia Injury
2
Liposome-mediated Cas13a Delivery for EV Fusion
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The liposome complex was prepared with Lipofectamine CRISPRMAX Cas9 Transfection reagent (ThermoFisher Scientific) by a brief modification of the manufacturer's instructions. Briefly, complex A, including 100 nm LwaCas13a, 100 nm NC or miR‐21‐5p crRNA, 20U RNase inhibitor, and 40 nm FAM‐tagged quencher reporter, was prepared in 1X Cas13a reaction buffer. Additionally, complex B was prepared by gently mixing transfection reagent with 1X Cas13a reaction buffer in an equal volume of complex A. The complexes A and B were gently mixed and incubated at RT for 15 min. Next, the AF647‐labeled EVs diluted in 1X Cas13a buffer were introduced into the complex and incubated at 37 °C for 30 min. After incubation, the fusion complex was mounted on the TPFE‐printed glass slide (Electron Microscopy Sciences) and analyzed using an upright fluorescent microscope (Zeiss).
3
Immunofluorescence Staining of Muscle Cells
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Cells and myofibers were fixed in 4% PFA for 5 min, permeabilised 5 min in 0.5% Triton-X100 (Sigma-Aldrich) and blocked in 10% normal goat-serum (Gibco) for 30 min at RT. Cells and fibres were then incubated with primary antibodies (Chick GFP, Abcam13970,1/2000; mouse Pax7, DHSB, 1/40; rabbit Myod, Santa Cruz, 1/200; mouse Myogenin (F5D), DHSB, 1/40) overnight at 4 °C. Samples were washed with 1X PBS three times and incubated with Alexa-conjugated secondary antibodies (Life Technologies, 1/1000) and Hoechst (Life Technologies, 1/10000) for 45 min at RT. EdU staining was chemically revealed using the Click-iT Plus kit according to manufacturer’s recommendations (Life Technologies, C10640). Images were acquired using an upright fluorescent microscope (Zeiss).
4
Quantification of DNA Damage Response Foci
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NSCLCs were seeded onto Lab-Tek II Chamber Slides (Thermo Fisher) and 24 hours later pretreated with JIB-04 or DMSO for 4 h. Then cells were exposed to a total dose of 2 Gy (γH2AX and 53BP1) or 10 Gy (RAD51 and DNAPKcs p-T2609) radiation. Cells were fixed in 4% formaldehyde/PBS for 15 min, permeabilized with 0.5% Triton X-100 for 15 min on ice, and blocked with 5% bovine serum albumin in PBS for 1 h. The slides were incubated with an antibody against phospho-Histone γH2AX (1:1000, 3 h at room temperature), 53BP1 (1:500, 3 h at room temperature), Rad-51 (1:500, 48 h 4C) or DNAPKcs p-T2609 (1:500, 48 h 4C). Alexa Fluor 488–conjugated goat anti-Rabbit, Alexa Fluor 555–conjugated goat anti-mouse or rhodamine red–conjugated goat anti-mouse secondary antibodies were used (1:1000, 1h at room temperature). Slides were mounted in a Vectashield mounting medium containing 40,6-diamidino-2-phenylindole (DAPI). Cells were imaged on a Zeiss upright fluorescent microscope. Foci counting was performed on the resulting images using the CellProfiler (CellProfiler.org ) open-source cell image analysis software (algorithm available upon request) in a blinded fashion. Quantification was validated in several cases manually with ImageJ in a blinded fashion by two independent investigators.
5
Quantification of DNA Damage Response Foci
6
Antibody-Functionalized EV Detection on Gold Surfaces
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Antibody immobilization on gold microdisk arrays or plain gold was conducted, as described previously.[32 ] Briefly, IgG isotype control (14‐4714‐82, eBioscience), CD63 (215‐820, Ancell) or EpCAM (MA5‐12436, Invitrogen) antibodies, respectively diluted by 1:50, 1:100 or 1:20 in 10 mm phosphate buffer, were treated on the surface for 1 h. After washing out the non‐bounded antibodies with PBS, 10% BSA in PBS was treated for blocking. Next, the AF647‐labeled EVs derived from cell cultures (OV90 and TiOSE4) or clinical plasma samples were incubated for 1 h. The complex for the EV‐miRNA detection system was then introduced and incubated at 37 °C for 1 h. After washing with PBST, the fluorescent signal was obtained by an upright fluorescent microscope (Zeiss).
7
Quantifying Organoid Tissue Formation
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Imaging was performed on mounted cryosections/whole-organoids using a Zeiss upright fluorescent microscope. CellProfiler was used to segment and identify immunopositive nuclei in fluorescently-labelled cryosection images. This was divided by the total number of nuclei as ascertained by visualisation with Hoechst. Organoid growth was assessed by measuring diameter using ImageJ and assessed for significance through two-way ANOVA. Formation of polarized neuroepithelial tissue was quantified in blinded samples and subject to statistical testing using one-way ANOVA.
8
Visualization of DNA Damage Foci
9
Alizarin Complexone Bone Labeling
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Mice were injected intraperitoneally with 5 μl/g body weight of a 5 mg/ml Alizarin complexone (VWR 20118.081) made up in 2% NaHCO3 in PBS. Calvaria were dissected 24 h after injection and imaged using a Zeiss upright fluorescent microscope with a 550 nm filter.
10
Constructing and Validating MGMT Expression Plasmids
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Plasmids were constructed via standard recombinant cloning techniques and all changes were verified by DNA sequencing. Human MGMT cDNA (MGMT [untagged]-human O6-methylguanine-DNA methyltransferase_wild type; OriGene [cat # SC322190]) was amplified via PCR and cloned into pcDNA3.1-Flag vectors. The pcDNA3.1-Flag-MGMT vector was generated using primers 5ʹ- TGACAAGCTTATGCTGGGACAGCCCGCGCCCCTAGAA-3ʹ (HindIII) and 5ʹ- TCCGAATTCCTAGTTTCGGCCAGCAGGCGGGGAGCCCGA-3ʹ (EcoRI). MGMT was then inserted between the HindIII and EcoRI restriction sites in pEGFP-N1 vectors. Transfection with these plasmids was performed using Lipofectamine™ 3000 (Thermo Fisher Scientific) in accordance with the manufacturer’s guidelines. MGMT overexpression levels were determined by immunoblot analysis and imaging using a Zeiss upright fluorescent microscope.
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