Multiplex immunohistochemistry/immunocytochemistry (mIHC/mICC) was performed as previously described [56 (link)]. Paraffin-embedded luteal sections were first dewaxed and subjected to microwave treatment for antigen retrieval. The luteal cells were seeded on glass coverslips (Solarbio, Beijing, China). The endogenous peroxidase activity was quenched by hydrogen peroxide. Slides were then blocked and incubated with primary antibody (Table 1 , APN, T-Ca, AdipoR1, AdipoR2, P-AMPK, AMPK, HSD3B, STAR) at 4 °C overnight. The negative controls (NC) were established by replacing the primary antibody with normal serum [57 (link)]. Sections were further incubated with a standard streptavidin–biotin–peroxidase complex (Boster Biological Technology, Wuhan, China) at room temperature. Then, a chromogenic working solution was added and optimized by employing a triplex (AF488, Cy3 and Cy5, ATT, Dallas, TX, USA) and were finished with DAPI. Image capture was performed with a Zeiss LSM900 (Zeiss, Jena, Germany).
Glass coverslips
Manufactured by Solarbio
Sourced in China
Glass coverslips are thin, transparent sheets of glass used in various scientific and laboratory applications. They serve as a platform for mounting and protecting samples for microscopic examination.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 900, by Zeiss (1 mentions)
Streptavidin biotin peroxidase complex, by Boster Bio (1 mentions)
Fat free milk, by Sangon (1 mentions)
Triton x 100, by Sangon (1 mentions)
Red tris nta, by NanoTemper (1 mentions)
Hochest 33342, by Beyotime (1 mentions)
Axr confocal microscope, by Nikon (1 mentions)
Edu assay kit, by RiboBio (1 mentions)
Pkh26 fluorescent dye, by Merck Group (1 mentions)
Fluorescence microscope, by Leica (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Tcs sp8 confocal microscope, by Leica (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Solarbio (1 mentions)
Paraformaldehyde (pfa), by Solarbio (1 mentions)
Tcs sp8 laser scanning microscope, by Leica (1 mentions)
Antifade mounting medium, by Solarbio (1 mentions)
6 protocols using glass coverslips
1
Multiplex Immunohistochemistry of Luteal Cells
2
Visualizing IL-17A Binding in RAW264.7 Cells
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RAW264.7 cells were plated on the glass coverslips (Solarbio) in 24-well plates for 1 day before the experiment. Cells were incubated with the peptide mixture and red fluorescent molecule RED-tris-NTA (Nanotemper) labled-IL-17A for 6 h. Cells were then fixed with 4% paraformaldehyde for 30 min, washed three times, permeabilized, and blocked with 0.3% Triton X-100 (Sangon Biotech) and 3% fat-free milk (Sangon Biotech) for 1 h. After the incubation with the primary antibody at 4 °C overnight, cells were then incubated with the appropriate secondary antibody and Hochest 33342 (Beyotime) at 37 °C for 30 min. Fluorescence images were captured by Nikon AXR confocal microscope using a Plan APO ×60, 1.42-NA oil objective with 405-nm violet laser, 488-nm blue laser, 561-nm green laser, and 640-nm red laser. Image analysis was performed with NIS-Elements Viewer and ImageJ software.
3
Cell Proliferation Quantified by EdU Assay
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24-well plates were used to seed cells on glass coverslips (Solarbio, Beijing, China) for 24h. EdU solution was added to the plate and incubated the cells for 2h. Then 4% paraformaldehyde and 0.5% Triton X-100 were used to fix and infiltrate the cells. Finally, Apollo and hoechst33342 were utilized to stain for 30min. The EdU assay kit (RiboBio, China) was applied to assess cell proliferation.
4
Exosome Internalization in RAW 264.7 Cells
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We labeled USC-Exos with PKH26 fluorescent dye (Sigma-Aldrich) to observe whether RAW 264.7 cells can internalize exosomes. Briefly, 1.0 mL of Diluent C was added to 800 μg of exosome solution. Then, 4 μL of PKH26 dye was added to the mixture and cocultured for 5 min at room temperature in the absence of light. The labeled exosomes were then centrifuged at 100,000 rpm for ~25 min at 4°C, and the exosome pellet was suspended in PBS and used for uptake experiments. RAW 264.7 cells were seeded into glass coverslips (Solarbio) and cultured for 24 h until reaching 80–90% density. We then cocultured 25 μL (100 μg) of PKH26-labeled USC-Exos with RAW 264.7 cells in serum-free DMEM culture medium for 4, 12 and 24 h. After the cells were fixed in 4% paraformaldehyde (PFA) for 20 min and permeabilized with 0.1% Triton X-100, the cells were stained with phalloidin Alexa Fluor 488 (A12379; Invitrogen, USA) for 1 h at room temperature. Images were examined and captured under a fluorescence microscope (Leica, Germany).
5
Immunofluorescence Imaging of Cultured Cells
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The cells were cultured on glass coverslips (Solarbio, China) for the indicated treatment concentrations, then fixed in 4% paraformaldehyde and PBS for 30 min, and then permeabilized with 0.25% Triton X-100 according to the specific requirement. The nuclei were stained with DAPI (Invitrogen, USA). The images were taken under a fluorescence microscope or the Leica TCS SP8 confocal microscope.
6
Multimodal Analysis of TGF-β Signaling
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Cells were seeded in 24-well plate with glass coverslips (Solarbio), fixed with 4% (w/v) paraformaldehyde (Solarbio) for 10 min at room temperature, washed with 1× PBS, 70% ethanol for 1 hr, and washed with Stellaris Buffer A (Biosearch). Then cells were incubated with Stellaris FISH probes which were diluted by Stellaris FISH Hybridization Buffer (1:100) at 37℃ overnight. Cells were next washed with Buffer A for 30 min at 37℃ and B for 5 min at room temperature. Primary antibodies TGF-β (dilution, 1:100), TGFBR1 (dilution, 1:1000), and SMAD1/5/9 (dilution, 1:500) with 5% BSA in PBS were, respectively, incubated with cells for 2 hr at room temperature. Then, cells washed three times with PBS and incubated with fluorescein isothiocyanate (FITC)-labeled goat anti-rabbit/mouse secondary antibody (Beyotime) for 30 min at room temperature. Finally, coverslips were mounted onto slides with antifade mounting medium supplemented with DAPI (Solarbio). Confocal images were acquired using a Lecia TCS SP8 laser scanning microscope.
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