Lp18:S cells were cultured in MRS for 12 h, washed with PBS twice, and collected by centrifugation at 8000 ×g for 2 min. The pellet was mixed with a primary antibody (anti-HA tag rabbit polyclonal antibody, 1:100, Proteintech, USA) at 4 °C overnight, followed by three washed with PBS. Then, the bacteria were incubated with a FITC-conjugated secondary antibody (FITC-conjugated goat anti-rabbit IgG, 1:3000, Zsbio, China) at 37 °C for 30 min. After washing 4 times with PBS, 3 μL cells were fixed on a clean coverslip in the dark, followed by staining with 5 μL Antifade Polyvinylpyrrolidone Mounting Medium (Beyotime, Shanghai, China) on the coverslip. Thereafter, the coverslip was mounted on a clean slide, and the samples were examined by fluorescence microscopy.
Antifade polyvinylpyrrolidone mounting medium
Manufactured by Beyotime
Sourced in China
Antifade Polyvinylpyrrolidone Mounting Medium is a laboratory reagent used to preserve and protect fluorescent signals in microscopy samples. It is designed to prevent the fading or quenching of fluorescent dyes and proteins, enabling long-term storage and visualization of labeled specimens.
Automatically generated - may contain errors
Lab products found in correlation
Anti ha tag rabbit polyclonal antibody, by Proteintech (1 mentions)
Fitc conjugated goat anti rabbit igg, by ZSGB-BIO (1 mentions)
Dmi400b, by Leica camera (1 mentions)
P0102, by Beyotime (1 mentions)
P0123, by Beyotime (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (1 mentions)
Lipo8000 transfection reagent, by Beyotime (1 mentions)
Bodipy, by Thermo Fisher Scientific (1 mentions)
Fluorescence microscope, by Leica camera (1 mentions)
Alexa fluor 488 conjugated goat anti mouse secondary antibody, by Beyotime (1 mentions)
Cy3 conjugated goat anti rabbit secondary antibody, by Beyotime (1 mentions)
Confocal microscope, by Leica camera (1 mentions)
Chemiluminescence detection kit, by Merck Group (1 mentions)
Integrin β3, by Abcam (1 mentions)
Glyceraldehyde 3 phosphate dehydrogenase gapdh, by Proteintech (1 mentions)
Hematoxylin eosin, by Beyotime (1 mentions)
P enos, by Abcam (1 mentions)
Integrin αv, by Abcam (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
6 protocols using antifade polyvinylpyrrolidone mounting medium
1
Immunofluorescence Localization of HA-tagged Cells
2
Immunofluorescence Staining of Nrf2 in Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
After being treated as described in method 2.6, cells were washed using 0.5% PBST and fixed with paraformaldehyde of 4% for 10 min, followed by being washed using 0.5% PBST three times and blocked using immunol staining blocking buffer (P0102, Beyotime Institute of Biotechnology, Shanghai, China) for 1.2 h. Then the cells were incubated with Nrf2 rabbit anti-human primary antibody (1:100 dilution) at 4°C overnight, followed by being kept at room temperature for 1 h and washed using 0.5% PBST three times. The cells were subsequently incubated with FITC labeled goat anti-rabbit secondary antibody (1:200 dilution) at 37°C in a humidified incubator for 1.5 h, followed by being washed using 0.5% PBST three times. Finally, cells were stained with Hoechst 33258 of 20 μM for 5 min, followed by being washed using 0.5% PBST three times and adding antifade polyvinylpyrrolidone mounting medium (P0123, Beyotime Institute of Biotechnology, Shanghai, China). Then the slices were observed via a fluorescence microscope (Leica DMI400B).
3
Cloning and Expression of U1-70K Fragments
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Three DNA fragments (LC1, LC2, and LC) and full-length U1-70K were amplified using six primers (table S2). LC1 was cloned with U1-Nhe231f and U1-Sac308rnostop; LC2 was cloned with U1-Nhe317f and U1-Sac407rnostop; LC was cloned with U1-Nhe231f and U1-Sac407rnostop; full-length U1-70K was cloned with U1-Nhe1f and U1-Sac1314rnostop. The PCR fragment was gel-purified, digested by restriction endonucleases (Nhe I and Sac II), and cloned into the pCDNA3.1-EGFP vector (Nhe I and Sac II digested). The EGFP-fused proteins were expressed by N2a cells.
N2a cells were cultured in complete media [44.5% DMEM 1× with glucose (4.5 g/liter), 44.5% MEM Alpha 1×, 10% FBS, and 1% penicillin/streptomycin]. N2a cells were plated in 24-well plate with microscope coverglass (φ14 mm, CITOGLAS, China) on the bottom and were allowed to grow for 12 hours. The plasmids of pCDNA3.1-LC1-EGFP, pCDNA3.1-LC2-EGFP, or pCDNA3.1-LC-EGFP were transfected with the Lipo8000 Transfection Reagent (Beyotime Biotechnology, China) into the cells for protein expression. After 24 hours, 4% paraformaldehyde was used to fix the cells and cell nucleus was stained with 4′,6-diamidino-2-phenylindole (DAPI) (Beyotime Biotechnology, China) for 30 min at 37°C. The Antifade Polyvinylpyrrolidone Mounting Medium (Beyotime Biotechnology, China) was applied to the samples and mounted to the slide glass (CITOGLAS, China).
N2a cells were cultured in complete media [44.5% DMEM 1× with glucose (4.5 g/liter), 44.5% MEM Alpha 1×, 10% FBS, and 1% penicillin/streptomycin]. N2a cells were plated in 24-well plate with microscope coverglass (φ14 mm, CITOGLAS, China) on the bottom and were allowed to grow for 12 hours. The plasmids of pCDNA3.1-LC1-EGFP, pCDNA3.1-LC2-EGFP, or pCDNA3.1-LC-EGFP were transfected with the Lipo8000 Transfection Reagent (Beyotime Biotechnology, China) into the cells for protein expression. After 24 hours, 4% paraformaldehyde was used to fix the cells and cell nucleus was stained with 4′,6-diamidino-2-phenylindole (DAPI) (Beyotime Biotechnology, China) for 30 min at 37°C. The Antifade Polyvinylpyrrolidone Mounting Medium (Beyotime Biotechnology, China) was applied to the samples and mounted to the slide glass (CITOGLAS, China).
4
BODIPY Fluorescent Staining Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The staining solution containing 2 μm BODIPY (D3922; Thermo Fisher Scientific, Waltham, MA, USA) was prepared in PBS. After PA or OA : PA treatment, cells were incubated with 4% paraformaldehyde for 15 min. Next, the fixed cells were incubated with 3 mL of staining solution for 15 min at 37 ºC in the dark. After washing in PBS, a drop of antifade polyvinylpyrrolidone mounting medium (Beyotime, Jiangsu, China) with 4ʹ,6‐diamidino‐2‐phenylindole was added onto the slides. Images were obtained with a fluorescence microscope (Leica, Wetzlar, Germany).
5
Macrophage Polarization Immunostaining Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mφs on the climbing piece in 24-well culture plates were treated with or without bacterium infection or treated with relevant CM for 48 h. Then the cells were fixed with 4% paraformaldehyde for 20 min, washed with PBS, and permeabilized with 0.01% Triton X-100 for 10 min. After being washed with PBS, the cells were blocked with 10% goat serum for 30 min at room temperature and incubated with anti-CD86 monoclonal antibodies (Santa Cruz, USA) and anti-CD206 antibodies (Proteintech, China) at 4°C overnight. The next day, the cells were rinsed with PBS for clearing the primary antibody, and then incubated with Alexa Fluor 488-conjugated goat anti-mouse secondary antibody (Beyotime, China) or Cy3-conjugated goat anti-rabbit secondary antibody (Beyotime, China) at room temperature for 1 h in the dark, and then washed with PBS, and counterstained the nucleus with DAPI for 10 min. After washing again with PBS and mounting with antifade polyvinylpyrrolidone mounting medium (Beyotime, China), the fluorescent images were observed using confocal microscope (Lecia, Germany).
6
Implantation Regulation by Mifepristone and Progesterone
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mifepristone and progesterone were obtained from the pharmacy department of Yueyang Hospital of Integrative Traditional Chinese and Western Medicine; hematoxylin-eosin, Antifade Polyvinylpyrrolidone Mounting Medium, and nitric oxide (NO) assay kit were purchased from Beyotime (China); the antibodies ERα, ERβ1, PR, Integrin αV, Integrin β3, OPN, LIF, eNOS, and p-eNOS were purchased from Abcam (UK); vascular endothelial growth factor (VEGF) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were purchased from Proteintech (USA); Alexa Fluor 488 nm, Alexa Fluor 555 nm, Akt, p-Akt, and β-tubulin were purchased from CST (USA); Lycopersion Esculentum Lectin was purchased from Vector Laboratories (USA); DAPI was purchased from Sigma (USA); chemiluminescence detection kit was purchased from Millipore (USA).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!