Tissue sections of 5-μm thickness were cut from frozen glioblastoma specimens and processed for immunofluorescence. Sections were immunostained with rabbit anti-ATF5 (Abcam, ab60126) and mouse anti-IE (Virostat-Inc, 0841) antibodies and then stained with anti-rabbit IgG conjugated with CY3 (boster, BA1032), and anti-mouse IgG conjugated with FITC (boster, BA1101). Following repeated washes in PBS, nuclei were counterstained with Hoechst dye. The images were obtained using Olympus FV1200 fluorescencemicroscope.
Fv1200 fluorescence microscope
Manufactured by Olympus
Sourced in Japan
The FV1200 is a fluorescence microscope designed for high-resolution imaging of biological samples. It features a motorized stage, a range of objective lenses, and advanced imaging capabilities to capture detailed fluorescence images.
Automatically generated - may contain errors
Lab products found in correlation
Ba1032, by Boster Bio (2 mentions)
Ba1101, by Boster Bio (2 mentions)
Paraformaldehyde (pfa), by Biosharp (1 mentions)
Alexa fluor conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Flag tag (flag), by Merck Group (1 mentions)
5 protocols using fv1200 fluorescence microscope
1
Immunofluorescent Staining of Glioblastoma Tissues
2
Immunohistochemical Analysis of ZO-1 and TIMELESS
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Tissue specimens were fixed with 4% paraformaldehyde (Biosharp, China), paraffin-embedded, and cut into 5-μm-thick sections. Then, an UltraSensitive SP Immunohistochemistry (IHC) Kit (Maxim Biotech, Fuzhou, China) was used following the same steps as we did previously3 (link),4 (link). Sections were incubated with primary antibodies against ZO-1 (Abcam ab221547) and TIMELESS (Proteintech 14,421–1-AP) overnight at 4°C. Finally, an Olympus FV1200 fluorescence microscope (Olympus, Japan) was used to acquire images for analysis.
3
Immunofluorescence Assay of ATF5 and IE Proteins
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U87 and shATF5-U87 cells were maintained in serum free medium and were trypsinized and replated onto glass coverslips in 12-well tissue culture plates. After infection, cells on the glass coverslips were first fixed with buffered 4% paraformaldehyde, pH 7.4, for 30 min at room temperature and then permeabilized with 0.1 M PBS containing 0.2% Triton X-100 for 30 min and thereafter preincubated with 5% normal goat serum for 30 min prior to incubation with the primary antibodies at 4°C for overnight. The primary antibodies employed were against the antigens ATF5 (diluted 1:1000; Abcam) and immediate-early (IE) protein (diluted 1:100; Virostat-Inc). After thorough rinsing with 0.1 M PBS, slides were incubated with the secondary antibody conjugated CY3 (diluted 1:500; boster, BA1032) or FITC (diluted 1:200; boster, BA1101). Following repeated washes in PBS, nuclei were counterstained with Hoechst dye. To ensure antibody specificity, negative control slides were processed in the same manner, but omitting the primary antibodies. The images were obtained using Olympus FV1200 fluorescencemicroscope.
4
Immunofluorescence Assay for Subcellular Localization
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Cells grown on coverslips were fixed with 3% paraformaldehyde for 15 min at room temperature followed by permeabilization in 0.5% Triton X-100 solution for 30 s. After blocking with 3% milk at room temperature for 20 min, cells were incubated sequentially with primary antibodies and secondary fluorophore-conjugated antibodies. DAPI (Sigma-Aldrich, 1:10,000) was used to stain nuclear DNA. The following antibodies were used for IFA assays: YAP (sc-101199, Santa Cruz, 1:100), Flag (F3165, Sigma-Aldrich, 1:1,000), and Alexa Fluor–conjugated secondary antibodies (Thermo Fisher Scientific, 1:400). All the images were captured using a 60× oil immersion lens on an Olympus FV1200 fluorescence microscope and were further processed by ImageJ software.
5
FISH-based Quantification of Fn in Colon
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Microbial FISH was performed following the manufacturer’s instructions to assess the abundance of Fn in human colon tissue (Focobio, China). The sequence of the Fn targeting probe (FUS664; FITC-labeled) was 5’-CTT GTA GTT CCG C(C/T) TAC CTC-3’4 (link). Slides were photographed using the 400× lens on an Olympus FV1200 fluorescence microscope (Olympus, Japan). The average number of FUS664 probes per field of view was observed and calculated by three investigators who were blinded to the experimental protocol to evaluate Fn abundance. We defined Fn-negative, Fn-low, and Fn-high as samples with an average number of < 5, 5–20, and > 20 FUS664 probes visualized per field on average, respectively4 (link).
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