Cells were seeded into 8-well LabTek removable chamber slides (Thermo Scientific, 177402). After treatment, cells were fixed with 4% paraformaldehyde (PFA) for 15 min before permeabilization with 0.1% saponin in blocking buffer (1% fatty acid free BSA, 20 mM glycine in PBS) for 15 min at room temperature. For non-permeabilized TREM2 staining, saponin was omitted from all blocking and washing steps. Then, samples were incubated with primary antibodies (rabbit polyclonal anti-TREM2, Proteintech, 13483–1-AP, 1:100, overnight at 4 °C; rabbit polyclonal anti-GSDMD, Abbexa, abx340202, 1:250, 3 h at 37 °C), followed by 1-h incubation at room temperature with the secondary antibody Alexa Fluor 488-conjugated goat anti-rabbit IgG (Thermo Fisher Sci., A-11008, 1:400). From each well, 3 non-overlapping images from the top, middle and bottom areas were randomly taken using a Leica TCS SPE confocal laser scanning microscope (Leica Microsystems) with a 63 × /1.32–0.60 oil PH3 CS objective and a confocal pinhole set at 1 Airy unit.
Labtek removable chamber slides
Manufactured by Thermo Fisher Scientific
LabTek removable chamber slides are a type of laboratory equipment designed to facilitate cell culture and observation. They feature a removable chamber that allows for easy access and manipulation of cells during experiments. The slides provide a controlled environment for cell growth and analysis.
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Lab products found in correlation
Leica tcs spe confocal laser scanning microscope, by Leica Microsystems (4 mentions)
Alexa fluor 488 conjugated goat anti rabbit igg, by Thermo Fisher Scientific (2 mentions)
Lipofectamine p300, by Thermo Fisher Scientific (2 mentions)
Cellmask orange plasma membrane stain, by Thermo Fisher Scientific (2 mentions)
Donkey serum, by Bio-Rad (1 mentions)
Dapi containing solution, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 594, by Thermo Fisher Scientific (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
5 protocols using labtek removable chamber slides
1
Immunofluorescence Staining of TREM2 and GSDMD
2
Visualization of ASC Oligomer Formation
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To evaluate the presence of ASC oligomers, cells were transfected with the pLEX-MSC-ASC-GFP plasmid, a gift from Christian Stehlik (Addgene plasmid #73957; http://n2t.net/addgene:73957 ; RRID:Addgene_73957) [36 (link)]. Cells were seeded in 8-well LabTek removable chamber slides (Thermo Scientific, 177402) to 60%–70% of confluence. Transfections were conducted using Lipofectamine P300 (Invitrogen) in opti-MEM medium. Forty-eight hours after transfection, cells were exposed to the corresponding treatment. After treatment, cells were incubated with the CellMask™ Orange plasma membrane stain (1:10,000; Thermo Fisher Sci., C10045) and fixed with 4% PFA. The formation of ASC specks was assessed as the formation of reticulated structures, visualized with a Leica TCS SPE confocal laser scanning microscope (Leica Microsystems, Spain) using a 63 × /1.32–0.60 oil PH3 CS objective and a confocal pinhole set at 1 Airy unit. From each well, three non-overlapping images from the top, middle and bottom areas were randomly taken. The number of speck-positive cells was determined by manual counting using ImageJ [34 (link)].
3
Visualization of ASC Oligomer Formation
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To evaluate the presence of ASC oligomers, cells were transfected with the pLEX-MSC-ASC-GFP plasmid, a gift from Christian Stehlik (Addgene plasmid #73957; http://n2t.net/addgene:73957 ; RRID:Addgene_73957) [36 (link)]. Cells were seeded in 8-well LabTek removable chamber slides (Thermo Scientific, 177402) to 60%–70% of confluence. Transfections were conducted using Lipofectamine P300 (Invitrogen) in opti-MEM medium. Forty-eight hours after transfection, cells were exposed to the corresponding treatment. After treatment, cells were incubated with the CellMask™ Orange plasma membrane stain (1:10,000; Thermo Fisher Sci., C10045) and fixed with 4% PFA. The formation of ASC specks was assessed as the formation of reticulated structures, visualized with a Leica TCS SPE confocal laser scanning microscope (Leica Microsystems, Spain) using a 63 × /1.32–0.60 oil PH3 CS objective and a confocal pinhole set at 1 Airy unit. From each well, three non-overlapping images from the top, middle and bottom areas were randomly taken. The number of speck-positive cells was determined by manual counting using ImageJ [34 (link)].
4
Immunofluorescence Staining of TREM2 and GSDMD
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Cells were seeded into 8-well LabTek removable chamber slides (Thermo Scientific, 177402). After treatment, cells were fixed with 4% paraformaldehyde (PFA) for 15 min before permeabilization with 0.1% saponin in blocking buffer (1% fatty acid free BSA, 20 mM glycine in PBS) for 15 min at room temperature. For non-permeabilized TREM2 staining, saponin was omitted from all blocking and washing steps. Then, samples were incubated with primary antibodies (rabbit polyclonal anti-TREM2, Proteintech, 13483–1-AP, 1:100, overnight at 4 °C; rabbit polyclonal anti-GSDMD, Abbexa, abx340202, 1:250, 3 h at 37 °C), followed by 1-h incubation at room temperature with the secondary antibody Alexa Fluor 488-conjugated goat anti-rabbit IgG (Thermo Fisher Sci., A-11008, 1:400). From each well, 3 non-overlapping images from the top, middle and bottom areas were randomly taken using a Leica TCS SPE confocal laser scanning microscope (Leica Microsystems) with a 63 × /1.32–0.60 oil PH3 CS objective and a confocal pinhole set at 1 Airy unit.
5
Immunofluorescence Staining of dCas9-Fusion Proteins
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For immunofluorescence staining, HEK-293T cells transiently transfected with the dCas9-fusion constructs were seeded onto Lab-Tek removable chamber slides (ThermoFisher, Cat. No. 154534 PK) 24 h after transfection and induced with Dox (50 ng/L) 8 h later. After 24 h of Dox induction, the cells were fixed using 4% paraformaldehyde (Sigma, Cat. No. 1.00496), permeabilized with 0.1% Triton X-100 (Sigma, Cat. No. T8787), and blocked with PBS buffer containing 5% donkey serum (Bio-Rad, Cat. No. C06SBZ). The slides were incubated at 4 • C overnight with mouse anti-Cas9 primary antibody (Cell Signaling Technology, Cat. No. 14697) diluted in blocking solution (1:500). After incubation, the slides were washed with blocking solution and incubated for 1 h at room temperature in the dark with donkey anti-mouse Alexa Fluor 594 (1:250; ThermoFisher, Cat. No. A-21203). Finally, the slides were washed with blocking solution and mounted with DAPI-containing solution (ThermoFisher, Cat. No. 00-4959-52). The cells were imaged with a SP8 Falcon system Leica microscope and the images were processed using ImageJ software.
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