Following stimulation, BMDMs were fixed in 4% paraformaldehyde for a period of 10 mins. The fixed cells were washed in PBS prior to permeabilization in 0.1% or 0.3% Triton-X in PBS for staining of PIEZO1P1-tdT or NFκB/STAT6, respectively. Following additional PBS washes the cells were blocked in 2% BSA prior to being incubated with primary antibodies for 1 h at room temperature or overnight at 4 °C (see Supplementary table 1 for list of antibodies used). The cells were then repeatedly washed with 2% BSA and incubated with secondary antibodies in 2% BSA, for 1 h at room temperature. After repeated washing with 2% BSA, the cells were incubated with Alexa Fluor 488 phalloidin (Fisher Scientific), diluted 1:100 in PBS, and Hoechst (Invitrogen), diluted 1:2000 in PBS, for 30 min at room temperature. The cells were thoroughly washed with PBS, before being mounted onto a glass slide and imaged using a Zeiss LSM700 confocal microscope or an Olympus Fluoview FV3000 confocal laser scanning microscope. Approximately 50 cells in each condition were outlined per experiment and the mean intensity or total intensity was computed for each cell using ImageJ.
Lsm700 confocal microscope
Manufactured by Olympus
The LSM700 is a confocal microscope designed for high-resolution imaging of samples. It utilizes laser scanning technology to capture detailed images of microscopic structures. The core function of the LSM700 is to provide a precise and versatile imaging platform for research and analysis applications.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 594 conjugated antibody, by Cell Signaling Technology (2 mentions)
Ix71 research inverted system microscope, by Olympus (2 mentions)
Dp73 17mp color camera, by Olympus (2 mentions)
Alexa fluor 488 phalloidin, by Thermo Fisher Scientific (1 mentions)
Hoechst, by Thermo Fisher Scientific (1 mentions)
Fluoview fv3000 confocal laser scanning microscope, by Olympus (1 mentions)
4 protocols using lsm700 confocal microscope
1
Confocal Imaging of PIEZO1 and NFκB/STAT6
2
Immunofluorescence Staining of Phosphorylated Histone H3
3
Immunofluorescence Staining of Phosphorylated Histone H3
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Cytospin slides were fixed in 4% paraformaldehyde for 1 h, permeabilized in 0.25% Triton X-100 in PBS, blocked in 1% BSA and 2% FBS in PBS, followed by staining with AlexaFluor 488-conjugated anti-p-H3 antibody (22 (link)). Slides were mounted using Vectashield containing DAPI. Images were captured using a Zeiss LSM 700 confocal microscope or Olympus IX71 Research Inverted System Microscope with a DP73;17MP Color Camera.
For double staining for EdU and p-H3, cytospin slides were prepared after pulse labeling with EdU, followed by immunofluorescence staining for p-H3 using secondary AlexaFluor 594-conjugated antibody (Cell Signaling).
For double staining for EdU and p-H3, cytospin slides were prepared after pulse labeling with EdU, followed by immunofluorescence staining for p-H3 using secondary AlexaFluor 594-conjugated antibody (Cell Signaling).
4
Confocal Imaging of Neuronal Punctae
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Confocal z-stacks were acquired on a Zeiss LSM700 confocal microscope using the 63×/1.40 NA oil objective or Olympus FV3000 confocal microscope using the 60×/1.5 NA oil High Resolution objective. Sequential frame acquisition was set to acquire an average of 10 planes per stack at 16 bits and a minimum of 1024 × 1024 resolution. Channel gain settings were optically adjusted to minimize saturation of punctae and were maintained across experimental groups. Unmodified images were used for all analyses that were done blind and linear scaling was applied on images only for presentation purposes using ImageJ software. Fluorescent signal on the single planes from a stack, quantification of punctae number and punctae integrated fluorescence intensity over area measurements were performed with ImageJ software. Untreated and treated cells from the same culture preparation were always compared. For each experimental group an average of > 200 μm dendrite was quantified for 8–12 images per experiment in duplicate and repeated for a total of three or four independent experiments.
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