Cells (BMOL1.2, BMOL-TAT) were plated at ~70% sub-confluency in chamber slides (Sarstedt). Once reaching confluence, following a rinse in PBS, cells were fixed in 10% neutral buffered formalin for 20 min at RT. Following washing (3 x 5 min, 1x PBS), permeabilisation (0.5% (v/v) TX-100/PBS, 20 min RT), and another wash, blocking was performed using Odyssey buffer (LI-COR) for 1 h, RT. Primary antibodies were incubated for 2 h at RT and are listed above, excepting Osteopontin (AF808, R&D). Fluorescent signal was detected using secondary antibodies or direct conjugation to CF dyes, as above.
Chamber slides
Manufactured by Sarstedt
Sourced in Germany
Chamber slides are a type of laboratory equipment used for various microscopy applications. They provide a contained environment for samples, enabling observation and analysis under a microscope. The core function of chamber slides is to hold and secure samples in a controlled manner, facilitating the examination and study of biological or material specimens.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 710 confocal microscope, by Zeiss (3 mentions)
Lsm 700 microscope, by Zeiss (2 mentions)
Osteopontin af808, by R&D Systems (1 mentions)
Odyssey buffer, by LI COR (1 mentions)
Anti il 34 antibody, by Abcam (1 mentions)
Prolong gold antifade reagent with dapi, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
5 protocols using chamber slides
1
Immunofluorescent Staining of Cultured Cells
2
Immunofluorescent Staining of IL-34 in Caco-2 Cells
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Immunofluorescent staining was performed on Caco-2 cells cultured on chamber slides (Sarstedt) that were fixed in acetone followed by blocking with 10% (v/v) normal goat serum in PBS. Anti-IL-34 antibody (catalogue number AB-75723; Abcam) was added, and slides were incubated overnight at 4°C. chamber slides were incubated with secondary goat anti-rabbit IgG conjugated with Alexa Fluor® 488 (catalogue number A11034; Invitrogen), diluted in blocking buffer. Sections stained with the secondary antibody alone were used as negative controls. Sections were mounted in ProLong Gold antifade reagent with DAPI (Invitrogen). Slides were scanned on a LSM710 confocal microscope (Zeiss) in single-photon mode.
3
Radiation-Induced DNA Damage Quantification
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Cells (about 5·104) were seeded in 500 μl medium per well in 8-well Chamber Slides (Sarstedt, Nümbrecht, Germany) 20–24 h before irradiation. Cells were treated with PI-103 for 3 h prior to radiation with a single dose of 2 Gy. After irradiation, cells were kept in CGM for the indicated time until fixation. At different time points (0 min, 10 min, 20 min, 30 min, 120 min, 240 min and 360 min) after irradiation, cells were washed with pre-warmed (37 °C) PBS and fixed for 10 min at room temperature with a PBS solution containing 4% formaldehyde (Thermo Scientific, Rockford, IL). Thereafter, the cells were stained simultaneously for γH2AX and 53BP1 as previously decribed [26 (link), 27 (link)]. Confocal fluorescence images were acquired with a Zeiss LSM 700 microscope. In each sample, computer-assisted γH2AX foci counting from 3D-CLSM image stacks [28 (link)] was performed in about 100 cells.
4
GLUT4 Immunodetection in SGBS Adipocytes
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For the in situ immunodetection of GLUT4, SGBS adipocytes were cultured on chamber slides (Sarstedt, Nümbrecht, Germany). At day 8 cells were fixed with 4% paraformaldehyde for 25 min. After a washing step with PBS, endogenous peroxidases were blocked with H2O2 (3% in methanol) for 20 min. Further washing steps with tap water and PBST followed before the blocking with goat serum (10% in PBST) for 1 h. The primary antibody against GLUT4 (1:300; Sigma Aldrich) was incubated at 4°C overnight. Afterwards the secondary HRP-conjugated antibody goat anti-rabbit (1:1; Dako) was applied for 90 min at room temperature. Detection was performed with 3,3'-diaminobenzidin and hematoxylin was used for nuclear counterstaining.
5
Quantifying DNA Damage Foci by Confocal Microscopy
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The primary and secondary antibodies are speci ed in the Supplementary Information.
γH2AX foci counting using confocal laser scanning microscopy (CLSM)
Cells (about 5•10 4 ) were seeded in 500 µl medium per well in 8-well Chamber Slides (Sarstedt, Nümbrecht, Germany) 20-24 h before irradiation. Cells were treated with PI-103 for 3 h prior to radiation with a single dose of 2 Gy. After irradiation, cells were kept in CGM for the indicated time until xation. At different time points (0 min, 10 min, 20 min, 30 min, 120 min, 240 min and 360 min) after irradiation, cells were washed with pre-warmed (37 °C) PBS and xed for 10 min at room temperature with a PBS solution containing 4% formaldehyde (Thermo Scienti c, Rockford, IL). Thereafter, the cells were stained for γH2AX essentially as previously decribed [21] (link). Confocal uorescence images were acquired with a Zeiss LSM 700 microscope. In each sample, computer-assisted γH2AX foci counting from 3D-CLSM image stacks [22] (link) was performed in about 100 cells.
γH2AX foci counting using confocal laser scanning microscopy (CLSM)
Cells (about 5•10 4 ) were seeded in 500 µl medium per well in 8-well Chamber Slides (Sarstedt, Nümbrecht, Germany) 20-24 h before irradiation. Cells were treated with PI-103 for 3 h prior to radiation with a single dose of 2 Gy. After irradiation, cells were kept in CGM for the indicated time until xation. At different time points (0 min, 10 min, 20 min, 30 min, 120 min, 240 min and 360 min) after irradiation, cells were washed with pre-warmed (37 °C) PBS and xed for 10 min at room temperature with a PBS solution containing 4% formaldehyde (Thermo Scienti c, Rockford, IL). Thereafter, the cells were stained for γH2AX essentially as previously decribed [21] (link). Confocal uorescence images were acquired with a Zeiss LSM 700 microscope. In each sample, computer-assisted γH2AX foci counting from 3D-CLSM image stacks [22] (link) was performed in about 100 cells.
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