To determine changes in autophagy, both WT HEK293- and mutant UQCRB-expressing cell lines were seeded at a density of 1.5 × 105 cells/well in 6-well plates and incubated overnight. The medium was then changed to serum-free DMEM (Gibco, Rockville, MD, USA) and incubated for 24 h. After starvation, the cell samples were fixed with 4% formaldehyde (Sigma-Aldrich), diluted in phosphate-buffered saline (PBS), and washed with 1× PBS three times. The cells were then treated with LC3 primary antibody (Abcam, ab48394) for 2 h. Nuclei were stained with Hoechst (Invitrogen, Grand Island, NY, USA). Images were obtained using an LSM980 confocal microscope at 400× magnification.
Lc3 primary antibody
Manufactured by Abcam
Sourced in United States, United Kingdom
The LC3 primary antibody is a protein-specific antibody that recognizes the LC3 protein. LC3 is a key autophagy-related protein involved in the formation of autophagosomes. The antibody can be used to detect and study the expression and localization of LC3 in various biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Formaldehyde, by Merck Group (1 mentions)
Hoechst, by Thermo Fisher Scientific (1 mentions)
Lsm 710, by Zeiss (1 mentions)
Alexa fluor 488 anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Fluorescence microscope, by Olympus (1 mentions)
Donkey anti rabbit secondary antibody, by Thermo Fisher Scientific (1 mentions)
Fluorescence microscope, by Leica (1 mentions)
Goat anti rabbit igg antibody, by Cell Signaling Technology (1 mentions)
Lsm 510 meta, by Zeiss (1 mentions)
Triton x 100, by Beyotime (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (1 mentions)
7 protocols using lc3 primary antibody
1
Autophagy Evaluation in UQCRB Mutant Cells
2
Autophagy Analysis via Immunofluorescence Imaging
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To study the influence of the miR-23a expression level on autophagy activity, immunofluorescence imaging of autophagy markers was employed. RAW264.7 macrophages were fixed with 4% formaldehyde for 15 min at room temperature and incubated in 5% Tris buffered saline with Tween-20 (TBS-T; pH 8.3) diluted non-fat dry milk for 1 h. Immunofluorescence staining was performed using Microtubule-associated protein light chain 3 (LC-3) primary antibody (1:100; Abcam, Cambridge, UK; cat. no. ab62720) incubation overnight at 4°C and subsequent secondary antibody (1:200; Alexa Fluor 488 anti-rabbit IgG; Thermo Fisher Scientific, Inc.; cat. no. A10235) incubation at room temperature for 1 h. Cells were incubated using DAPI (100 ng/ml) for nuclear staining at room temperature for 30 min. Images were obtained from a confocal laser scanning microscope (LSM710; Zeiss, Oberkochen, Germany) at magnification, ×600. LC3 puncta were quantified with Image-J 1.51 (National Institutes of Health, Bethesda, MD, USA) (15 (link)).
3
Immunofluorescence Imaging of LC3
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The tissue sections undergo dewaxing, rehydration, and antigen repair extraction. Then, LC3 primary antibody (1 : 200 dilution, Abcam) was used for tissue sections to be incubated at 4°C overnight, donkey anti-rabbit secondary antibody (1 : 200 dilution, Life Technologies) was incubated at 37°C for 45 min, the cell nucleus was stained with DAPI dye, and the tablet was sealed with fluorescent tablet. Photo by fluorescence microscope (Olympus).
4
Hippocampal Autophagosome Localization
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
LC3, also known as LC3B-I, is a microtubule-associated protein that localizes to and accumulates in autophagosomes. 18 (link) The hippocampi were cut into 20-μm coronal sections, and incubated overnight with LC3 primary antibody (Abcam, UK) at 4°C. After incubation with goat anti-rabbit IgG antibody (Cell Signaling Technology, USA) at room temperature for 2 h, nuclei were counterstained with 4 ′ ,6-diamidino-2-phenylindole and observed under a fluorescence microscope (Leica, Wetzlar, Germany).
5
Immunofluorescence Analysis of Autophagy Markers
6
Immunofluorescence Assay of LC3 in SiHa Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immunofluorescence of SiHa cells was performed as previously described (9 (link)). After fixation with 4% paraformaldehyde for 15 min at 37 °C, SiHa cells were permeabilized with 0.1% Triton X-100 (Beyotime, China) for 30 min, and blocked with goat serum for 15 min. Cells were incubated with primary LC3 antibody (1 µg/m, Abcam) overnight at 4 °C, and then with AlexaFluor® 647 conjugated secondary antibody (#4414, 1:2,000, CST, USA) at room temperature for 60 min. DAPI (Beyotime) was used to counterstain the cell nuclei. Immunofluorescence images were acquired with confocal microscopy (LSM 510 Meta, Zeiss, Oberkochen, Germany). LC3 puncta were counted from 10 random fields per slide.
7
Immunofluorescence Analysis of Autophagy Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immunofluorescence staining was performed using keratinocytes grown on coverslips. Cells were left untreated or treated with the indicated cytokines for 24 hours prior to addition of alpha toxin for 2 hours. Cells were then washed, fixed with paraformaldehyde and permeabilized briefly with .5% Triton X-100. After blocking with BSA, sections were incubated with primary LC3 antibody (Abcam; Cambridge, MA) for 2 hours at room temperature. Secondary Cy-3 antibody (Jackson labs; West Grove, PA) was added for 1 hour. Images were taken with a Leica Microscope at 40x magnification using SlideBook software.
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!