Cells were seeded on glass coverslips, fixed in freshly prepared ice-cold 4% paraformaldehyde in PBS at room temperature for 10 min, permeabilized in 0.5% Triton X-100 in PBS at room temperature for 10 min, and then blocked with 2% bovine serum albumin (BSA) plus 5% FBS in PBS at room temperature for 1 h. After washing in PBS, the cells were incubated with primary antibodies at room temperature for 1 h, followed by incubation with the corresponding secondary antibodies at room temperature for 1 h. The cells were then mounted with medium containing 4’6-diamidino-2-phenylindole (DAPI, Vector Laboratories), and the preparations were visualized with an Olympus BX51 fluorescence microscope and a Zeiss confocal LSM 768 microscope. The antibodies used for immunoflurescence assay are listed in Supplementary Table 2.
Bx51 fluorescence microscope
Manufactured by Zeiss
Sourced in Germany
The BX51 fluorescence microscope is a high-performance optical instrument designed for advanced microscopy applications. It features a sturdy, ergonomic design and is equipped with a powerful fluorescence illumination system, enabling detailed observation and analysis of fluorescently labeled samples.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (3 mentions)
710 confocal microscope, by Zeiss (3 mentions)
Confocal lsm 768 microscope, by Zeiss (2 mentions)
Ab167453, by Abcam (1 mentions)
A11008, by Thermo Fisher Scientific (1 mentions)
Driselase, by Merck Group (1 mentions)
Dmem 10, by Thermo Fisher Scientific (1 mentions)
Mem non essential amino acid, by Thermo Fisher Scientific (1 mentions)
Iscove modified dulbecco media (imdm), by Merck Group (1 mentions)
Zen 2009, by Zeiss (1 mentions)
Vascular endothelial growth factor (vegf), by Santa Cruz Biotechnology (1 mentions)
6 protocols using bx51 fluorescence microscope
1
Cell Fixation and Immunofluorescence Protocol
2
Immunohistochemical Analysis of mCherry Expression
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A rat was deeply anesthetized with pentobarbital and transcardially perfused with heparinized saline and subsequently with ice-cold 4% paraformaldehyde in 1 × PBS (pH 7.4). The brain was removed and post-fixed in 4% paraformaldehyde overnight at 4°C, followed by dehydration with 30% sucrose in 1 × PBS. Tissues were sectioned into 30-μm thick coronal sections with a cryostat at –20°C. Sections were blocked with 5% normal donkey serum in PBS containing 0.3% Triton X-100 and incubated overnight with primary antibodies: mouse or rabbit anti-mCherry antibody, 1:500 dilution (Abcam Cat# ab167453, RRID:AB_2571870 ). Sections were then rinsed and incubated with the goat anti-mouse or goat anti-rabbit Alexa Fluor-conjugated secondary antibody, 1:500 (Molecular Probes Cat# A-11008, RRID:AB_143165 or Cat# A-11004, RRID:AB_141371 ). The sections were then mounted on slides, dried and coverslipped with ProLong Gold anti-fade reagent. The stained sections were examined with an Olympus BX51 fluorescence microscope or a Zeiss 710 confocal microscope.
3
Moss Cell Fixation and Microscopy
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We followed the protocol described by Vidali et al. (2007) (link) with the following modifications: sonicated moss was cultured for 6–7 days on the BCDAT plate, containing 5 µM β-estradiol for RNAi induction and 20 µg/ml G418 to prevent contamination. Collected cells were preserved in a fixative solution (2% formaldehyde, 25 mM PIPES, pH 6.8, 5 mM MgCl2, 1 mM CaCl2) for 30 min and washed three times with PME buffer (25 mM PIPES, pH 6.8, 5 mM MgCl2, 5 mM EGTA). Following fixation, cells were mounted on 0.1%PEI (polyethyleneimine)-coated glass slides and subsequently incubated with 0.1% Triton X-100 in PME for 30 min and 0.2% driselase (Sigma-Aldrich) in PME for 30 min. Next, cells were washed twice in PME, twice in TBS-T buffer (125 mM NaCl, 25 mM Tris-HCl, pH 8, and 0.05% Tween 20) and mounted in 10 µg/mL DAPI in TBS-T for observation. Images were acquired with the Olympus BX-51 fluorescence microscope equipped with ZEISS Axiocam 506 Color and controlled by ZEN software. Fluorescent intensity was measured with ImageJ. Cytoplasmic background was subtracted.
4
Visualizing A. phagocytophilum Infection in RF/6A Cells
5
Indirect Immunofluorescence Assay Protocol
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The indirect immunofluorescent assays were conducted as described previously with minor modifications [15 (link)]. Briefly, CNE-1 and HNE-1 cells were seeded on glass coverslips in 24-well plates. Cells were fixed in freshly prepared ice-cold 4% formaldehyde for 20 min at room temperature, permeabilized with 1% Triton for 15 min and blocked with 5% bovine serum albumin (BSA) at room temperature for 2 h. After washing with PBS, cells were incubated with primary antibodies at 4°C for overnight. The next day, after washing with PBS, cells were incubated with the corresponding secondary antibodies at 37°C for 1 h. The nucleus was stained with 4′6-diamidino-2-phenylindole (DAPI, Vector Laboratories) for 3 min. and cells were visualized with an Olympus BX51 fluorescence microscope and a Zeiss confocal LSM 768 microscope. For BrdU labelling, cells were treated with BrdU for 30min, fixed with 70% ethanol at 4°C, and the rest steps were the same as the indirect immunofluorescence assay. The antibodies used were listed in Supplementary Table 4 .
6
Immunohistochemical Analysis of VEGF and CD31 in Tumor Tissues
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Tumor tissues were fixed in 4 % paraformaldehyde for 24 h at 4°C, and tissue paraffin sections (5 µm) were incubated overnight. Slides were immunostained with primary antibodies against vascular endothelial growth factor (VEGF; 1:200, Santa Cruz Biotechnologies), CD31 (1:200, Abcam), and DAPI (Vector Labs, Burlingame, CA, USA). Fluorescent imaging was performed with an Olympus BX51 fluorescence microscope (magnification of x200), and images were analyzed using ZEN 2009 software (Carl Zeiss, Jena, Germany).
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