Paraffin-embedded human brain tissue sections (2 men and 1 woman for both LB positive and negative cases) were deparaffinized and hydrated through a series of graded ethanol solutions followed by PBS. The sections were incubated in Citrate buffer (10 mM Trisodium citrate, 0.05% Tween-20, pH6.0) at 90°C for 10 min for antigen retrieval. After washing in PBS, tissues were incubated in PBS-T (0.3% Triton X-100 in PBS) for 10 min. Tissues were blocked in 5% normal goat serum for 30 min and incubated in primary antibody at 4°C overnight. Immunostaining was visualized with biotinylated secondary, followed by avidin-biotin (Vectastain Elite Kit, Vector Labs), and 3,3’-diaminobenzidine reaction as previously described [43 (link)]. For immunofluorescence analysis, immunostaining was visualized with either fluorescent secondary (1:250 dilution; Alexa Fluor 488 or 594, Molecular Probes). For immunofluorescence staining of cultured cells, cells were grown and transfected on chamber slides. After antigen retrieval, cells were stained using same methods as tissue staining. Slides were scanned using an AperioScan Scope CS (40×magnification; AperioTechnologies Inc.) and fluorescence images of representative areas were acquired on EVOS FL Digital Microscope (EMS), Olympus IX81-DSU Confocal Microscope (Olympus), or TCS SP2 AOBS Spectral Confocal Microscope (Leica).
Ix81 dsu confocal microscope
Manufactured by Olympus
Sourced in Japan
The IX81-DSU Confocal Microscope is a high-performance imaging system designed for advanced microscopy applications. It features a spinning-disk confocal system that enables fast, high-resolution imaging of live specimens. The microscope is equipped with multiple laser lines and a range of magnification objectives to accommodate various sample types and research needs.
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Lab products found in correlation
Vectastain elite kit, by Vector Laboratories (1 mentions)
Tcs sp2 aobs spectral confocal microscope, by Leica (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Live dead viability cytotoxicity kit for mammalian cells, by Thermo Fisher Scientific (1 mentions)
Vectashield with dapi, by Vector Laboratories (1 mentions)
Bx60 microscope, by Olympus (1 mentions)
5 protocols using ix81 dsu confocal microscope
1
Immunohistochemical Analysis of Paraffin-Embedded Human Brain Tissue
2
Quantifying Cilia-Associated Protein Levels
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Stained sections and cells were examined and imaged using an Olympus IX81-DSU confocal microscope fitted with a 60x water objective. All images were captured as z-stacks (0.5 μm steps). The number of ciliated mCherry and mCherry/dnKif3a-positive cells and clones derived from single cell sorting was determined by counting the number of DAPI-labeled nuclei and aa-tubulin-positive cilia in each field/z-stack of randomly selected 4–6 microscopic fields per coverslip (3–5 coverslips/cell line and one coverslip/clone). The number of ciliated cells was expressed as a percentage of the total number of DAPI-labeled nuclei for each field. The mean background-corrected fluorescence intensity per pixel of Gli3C signal associated with aa-tubulin-positive cilia (n = 40 per group from 3 coverslips) was quantified using Image J software.
3
Live/Dead Cell Assay Protocol
4
Immunofluorescence Staining of Cells
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Cells were fixed in 4% paraformaldehyde in PBS for 10 min and permeabilized with 0.5% triton for 10 min prior to incubation in blocking buffer (2% fetal calf serum/3% bovine serum albumin in PBS) for 45 min. Primary antibodies diluted in blocking buffer were incubated between 1 h and overnight and following extensive washing with 0.1% Triton-PBS were labeled with fluorescence-conjugated secondary antibodies (1:1000, Invitrogen Alexa Fluor molecular probes). Vectashield with DAPI (Vector Laboratories) was used to stain nuclei. Images were captured using an Olympus BX60 microscope or Olympus DSU-IX81 confocal microscope (Olympus).
5
Immunofluorescence Staining of Inner Ear Cells
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Cells were grown in 48-well plates. After the respective treatments, cells were washed twice with PBS and fixed with freshly prepared 4% paraformaldehyde at room temperature for 15 min. For inner ear tissue staining, inner ear tissue was horizontally sliced into 5-µm sections, which were mounted on glass slides. Antigen accessibility was increased by treatment with 2% Triton X-100 for 10 min at 37°C. The samples were then blocked with 3% BSA for 30 min. The samples and cells were incubated with the primary antibodies overnight at 4°C. After washing three times with PBS, samples and cells were stained with a secondary antibody Alexa Fluor 488 goat anti-mouse IgG (H+L) (1:500 dilution; cat. no. A11032; Thermo Fisher Scientific, Inc.) for 1 h at 37°C. The nuclei were counterstained with DAPI for 15 min. After each incubation, the samples and cells were washed thrice with PBS for 5 min each. The morphology of SGN cells and the inner ear tissues were observed and images were captured with an Olympus DSU-IX81 confocal microscope (Olympus, Tokyo, Japan).
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