We constructed a plasmid containing SEP068184 and N-terminal fused enhanced GFP protein and then transformed this plasmid into the ΔSEP068184 mutant strain. The plasmid pRADG was transformed into WT R1 strain as control. Cells were spread on a glass slide and imaged by an LSM 880 fluorescent microscope (ZEISS). The nucleoid and plasma membrane were stained with 4′,6-diamidino-2-phenylindole (Beyotime) and Dil (Beyotime), respectively.
Lsm 880 fluorescent microscope
Manufactured by Zeiss
Sourced in Germany
The LSM 880 is a fluorescent microscope designed by Zeiss. It is capable of high-resolution imaging of fluorescently labeled samples. The microscope utilizes laser-based excitation and detection to capture detailed images of biological specimens.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Beyotime (1 mentions)
Anti myc tag mab, by Cell Signaling Technology (1 mentions)
Coralite488 conjugated affinipure goat anti rabbit igg, by Proteintech (1 mentions)
P4707, by Merck Group (1 mentions)
Histogel specimen processing gel, by Thermo Fisher Scientific (1 mentions)
Vybrant cm dil, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
5 protocols using lsm 880 fluorescent microscope
1
Visualizing SEP068184 protein localization
2
Immunofluorescence Imaging Protocol
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Immunofluorescence was performed as previously described75 (link). Primary antibodies used were C20 antibody and anti-myc tag mAb (Cell Signaling, Danvers, MA). Secondary antibodies were CoraLite488-conjugated Affinipure Goat Anti-Rabbit IgG and CoraLite594-conjugated Affinipure Goat Anti-Mouse IgG (proteintech, Wuhan, China). Images were captured by LSM 880 fluorescent microscope (Carl Zeiss, Jena, Germany) and analyzed with ZEN software.
3
Cell Death Detection with TUNEL Assay
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The TUNEL staining was performed with the In Situ Cell Death Detection Kit (12,156,792,910) according to the manufacturers' instructions. The images were captured by the LSM 880 fluorescent microscope (Carl Zeiss) and analyzed with the image j software.
4
Immunofluorescence Staining of Organoids
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Organoids grown in Matrigel were initially fixed in 4% PFA (Millipore Sigma catalog no. 47608) for 1.5 h. After PBS washing, organoids were embedded in HistoGel Specimen Processing Gel (Thermo Fisher Scientific catalog no. HG-4000-012), processed with an automated tissue processor (Sakura Tissue-Tek VIP), and embedded into a paraffin block (Tissue-Tek TEC). Samples were sectioned at 4 μm onto poly-L-lysine coated slides (Sigma-Aldrich; catalog no. P4707) and air-dried at RT over-night for any subsequent immunofluorescence staining. All slides for fluorescence were deparaffinized and antigen retrieved in pH 6 citrate buffer for a total of 40 min. After protein blocking, nuclei were stained with DAPI (Abcam; catalog no. ab104139). Infiltrating PBMCs were pre-stained with Molecular Probes Vybrant CM-Dil as described above. Imaging was performed on a Zeiss LSM880 fluorescent microscope with Zen Black software.
5
Paraffin-embedded Organoid Staining
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Organoids grown in Matrigel were initially fixed in 4% PFA for 1.5 h. After PBS washing, organoids were embedded in Histogel, processed with an automated tissue processor (Tissue-Tek VIP), and embedded into a paraffin block (Tissue-Tek TEC). Samples were sectioned at 4 μm onto poly-L-lysine coated slides and air-dried at room temperature over-night for any subsequent immunofluorescence staining. All slides for fluorescence were deparaffinized and antigen retrieved in pH 6 citrate buffer for a total of 40 min. After protein blocking, nuclei were stained with DAPI (Sigma). Infiltrating PBMCs were pre-stained with RFP as described previously. Imaging was performed on a Zeiss LSM880 fluorescent microscope with Zen Black software.
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