NPCs were cultured on different microspheres, then five groups were assigned in this part like those described above. There are three parallel samples of each group. They were fixed in 4% paraformaldehyde and then permeabilized with 0.3% Triton X-100 in PBS for 10 min after a 5-days culture. The microspheres were then soaked in Immunol Staining Blocking Buffer (Beyotime, Shanghai, China) for 1 h to block nonspecific binding. After incubation with 1:100 diluted anti-COX2 or 1:200 diluted anti-Col II antibodies at 4 °C overnight, Alexa Fluor® 488 antibodies (1:1000, Abcam Cambridge, UK) were used for fluorescent labeling. The nuclei were stained with DAPI and observed by the inverted fluorescence microscope. The primary antibodies are listed in Table S2 .
Alexa fluor 488 antibody
Manufactured by Abcam
Sourced in United Kingdom, United States
Alexa Fluor 488 antibody is a fluorescent dye-labeled antibody used for detection and visualization applications in biological research. It is a bright, green-fluorescent dye that can be conjugated to various antibodies, providing a sensitive tool for immunofluorescence, flow cytometry, and other fluorescence-based assays.
Automatically generated - may contain errors
Lab products found in correlation
Mitotracker red, by Thermo Fisher Scientific (3 mentions)
Confocal fluorescence microscope, by Leica (2 mentions)
Alexa fluor 594 antibodies, by Abcam (2 mentions)
Paraformaldehyde (pfa), by Biosharp (2 mentions)
Lsm 880, by Zeiss (2 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Fluo 4 am, by Thermo Fisher Scientific (2 mentions)
Immunol staining blocking buffer, by Beyotime (1 mentions)
Cd3 fitc, by Abcam (1 mentions)
Rabbit anti icam 1, by Abcam (1 mentions)
Alexa fluor 594 phalloidin, by Thermo Fisher Scientific (1 mentions)
Af6000, by Leica (1 mentions)
Alexa fluor594 antibody, by Abcam (1 mentions)
Tcs sp8 x, by Leica (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Roche (1 mentions)
Mouse anti neun, by Merck Group (1 mentions)
Rabbit anti gfap glial fibrillary acidic protein, by Merck Group (1 mentions)
Cm1900, by Leica (1 mentions)
Thioflavin s, by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (1 mentions)
19 protocols using alexa fluor 488 antibody
1
Immunofluorescence Analysis of NPC Differentiation
2
Immunohistochemical Analysis of Mouse Brain
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Anesthetized mice were fully perfused with PBS and 4% PFA (paraformaldehyde) and the brains were isolated and fixed with PFA. Before staining, the brain slices (10 μm thick) were washed with PBS three times and then incubated for 30 min in PBS containing 0.3% Triton X-100 and 3% serum. Later, the slices were incubated with primary antibodies for 12 h: rabbit anti-ICAM1 (1:100, Abcam) or mouse anti-A2AR (1:300, Wako). The sections were then washed with PBS and incubated for 2 h at room temperature with Alexa Fluor 488 antibodies (Abcam; 1:400). The slices were then washed and mounted on slides with VECTASHIELD mounting media (containing DAPI). For CD3 staining, the isolated CP tissues were fixed with 2.5% PFA for 30 min and then transferred to PBS. The following protocol was similar to the above procedure with rat anti-CD3 (1:100, CD3-FITC, Abcam). Images were acquired with a confocal microscope (LSM880, Zeiss). As described earlier [16 (link)], tissue slices were stained with hematoxylin–eosin.
3
Evaluating Osteogenic Differentiation Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Representative proteins (Runx2 and OPN) indicating osteogenic differentiation were evaluated in the BMSC, BMSC/BMP-4, BMSC/RAW, and BMSC/RAW/BMP-4 groups by fluorescence staining. Briefly, after culture of cells in conditioned medium for 12 days, scaffolds were fixed with 4% paraformaldehyde (Biosharp) for 15 min, permeabilized with 0.1% Triton-X for 30 min, and blocked with 5% BSA for 1 h. Samples were then incubated with primary polyclonal IgG rabbit anti-rat antibodies targeting Runx2 (1:500; Abcam) and OPN (1:200; Abcam) overnight at 4 °C and then with secondary goat anti-rabbit Alexa Fluor 594 antibodies (1:200; Abcam) for detection of Runx2 or secondary goat anti-rabbit Alexa Fluor 488 antibodies (1:500; Abcam) for detection of OPN for 2 h at room temperature. The cytoskeleton was stained with Alexa Fluor 488 for detection of Runx2 or Alexa Fluor 594 phalloidin (ThermoFisher) for detection of OPN for 2 h at room temperature. Nuclei were stained with DAPI for 5 min. A fluorescence confocal microscope (Leica, Germany) was used to acquire representative images. Image Pro Plus 6.0 software (NIH) was used to quantitatively analyze the fluorescence intensity of Runx2 and OPN in five different views for three independent scaffolds.
4
Quantitative Analysis of M1/M2 Macrophage Polarization
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The polarization of RAW264.7 cells in the RAW and RAW/BMP-4 groups was assessed by fluorescence staining to evaluate the expression levels of CCR7 (red, M1 marker) and CD206 (green, M2 marker). Briefly, after incubation in high-glucose DMEM for 2 and 5 days, scaffolds were fixed with 4% paraformaldehyde (Biosharp, Hefei, China) for 15 min, permeabilized with 0.1% Triton-X for 30 min, and blocked using 5% bovine serum albumin (BSA) for 1 h. Samples were incubated with M1 primary polyclonal IgG rabbit anti-mouse antibodies for CCR7 (1:100; Abcam) overnight at 4 °C and then with secondary goat anti-rabbit Alexa Fluor 594 antibodies (1:500; Abcam) for 2 h at room temperature. Next, samples were incubated with M2 primary polyclonal IgG mouse anti-mouse antibodies for CD206 (1:50; Abcam) overnight at 4 °C and then with secondary goat anti-mouse Alexa Fluor 488 antibodies (1:500; Abcam) for 2 h at room temperature. Finally, the nuclei were stained blue with 4′,6-diamidino-2-phenylindole, dihydrochloride (DAPI) for 5 min and observed with a fluorescence confocal microscope (Leica, Germany). Image Pro Plus 6.0 software (NIH) was used to quantitatively analyze the fluorescence intensities of CCR7 and CD206 for five different view regions from three independent scaffolds.
5
Immunofluorescence Analysis of RUNX1 and USP7-p53 Interaction
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
H9C2 cells were fixed in 4% paraformaldehyde (PFA) for 10 min and permeabilized using 0.2% Triton X-100. The cells were blocked using 10% bovine serum albumin (BSA) for 1 h and then incubated with anti-RUNX1 (1:200, Abcam) primary antibody at 4°C overnight. Further, the cells were washed with PBS 0.05% Tween (PBST) followed by incubation with Alexa Fluor594 antibody (1:1000, Abcam). After 1 h of incubation at 37°C, cell nuclei were stained with DAPI. Images were obtained by fluorescence microscope (Leica AF6000). The quantification of RUNX1 positive cells was performed using the ImageJ software.
The heart was dissected and then fixed in 4% PFA for 4 h. The heart sections (5 µm) were embedded in polyethylene glycol. USP7 (1:100; Cell Signaling Technology) and p53 (1:500; Cell Signaling Technology) antibodies were incubated to evaluate the interaction of USP7 and p53 in I/R rats. The sections were then incubated with Alexa Fluor 488 antibody (1:1000; Abcam) or Alexa Fluor594 antibody (1:1000, Abcam) for 2 h at 37°C. The nuclei were stained with DAPI. The images were visualized using Aconfocal microscopy (Zeiss 710).
The heart was dissected and then fixed in 4% PFA for 4 h. The heart sections (5 µm) were embedded in polyethylene glycol. USP7 (1:100; Cell Signaling Technology) and p53 (1:500; Cell Signaling Technology) antibodies were incubated to evaluate the interaction of USP7 and p53 in I/R rats. The sections were then incubated with Alexa Fluor 488 antibody (1:1000; Abcam) or Alexa Fluor594 antibody (1:1000, Abcam) for 2 h at 37°C. The nuclei were stained with DAPI. The images were visualized using Aconfocal microscopy (Zeiss 710).
6
Quantifying Endogenous LC3B Levels
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immunofluorescent staining was performed to determine the total endogenous levels of LC3B protein, as in our previous study [7 (link)]. Both the CA1 and DG regions were analyzed. In brief, we used a primary LC3B antibody (#3868; 1:200; CST) and a secondary Alexa-Fluor 488 antibody (#ab150077; 1:100; Abcam). Nuclei were counterstained with 4’,6-diamidino-2-phenylindole (1:5,000; Roche, Mannheim, Germany). Images were captured with a confocal fluorescence microscope (TCS SP8 X, Leica).
7
Immunohistochemical Analysis of Amyloid Plaques
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Animals were deeply anesthetized, killed, and perfused intracardially with normal saline and fixed with 4% paraformaldehyde (PFA). Brains were placed in 15% sucrose in PBS for 6-12 h, then 30% sucrose in PBS until equilibrated. Brain coronal sections (10 μm) were cut with a cryostat (Leica CM1900), sections were used for immunohistochemistry. Slices were washed with PBS and fixed with 4% paraformaldehyde, permeabilized with 0.05% Triton X-100, then pre-incubated in 1% BSA solution for blocking. Slices were incubated overnight at 4 °C with the primary antibody solution (rabbit anti-amyloid-β antibody, Abcam; 1:500), mouse anti-NeuN (1:100, Sigma), rabbit anti-glial fibrillary acidic protein (GFAP) (1:250, Sigma) on a shaker. After washing with PBS, the sections were incubated with the secondary antibody (goat anti-rabbit Alexa Fluor 488 antibody, Abcam; 1:1000; goat anti-mouse Alexa Fluor 488 antibody, Abcam; 1:1000) for 2 h at room temperature. Sections were mounted in the vectashield mounting medium with DAPI (Vector laboratories). For the detection of amyloid plaques, brain tissue sections were stained in 0.1% thioflavin-S (Sigma) and rinsed with 70% ethanol. The brain tissue sections were also stained with hematoxylin and eosin (H&E) to assess the histological damage.
8
Prostate Cancer Cell Imaging Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
PSMA (+) PC3-PIP or PSMA (−) PC3-Flu cells (0.2×106 cells/chamber in 500 µL of media) were seeded in 4-well chamber slides (Nunc Lab-Tek, Thermo Scientific) and grown to ∼90% confluency (24–48 h). Cells were then fed with fresh media containing 5D3-AF555 or 5D3(CC-MLN8237)3.2-AF555 (20 μg/mL) and incubated at 37°C for 1.5 h. Cells were washed twice with 1X DPBS and fixed using 4% PFA in PBS on ice for 10 min. After washing once with DPBS, cells were permeabilized with 0.1% Triton X-100 for 5 min and then blocked with 1.0% BSA/10% goat serum/0.3 M glycine in 0.1% PBS-Tween for 1 h. After a quick wash with 1X DPBS, cells were treated with centrosome marker, recombinant anti-pericentrin Alexa Fluor® 488 antibody (Abcam, Inc, 1:500 dilution), and anti-α-tubulin Alexa Fluor® 647 (Abcam, Inc. 1:250 dilution) for overnight at 4°C. Cell nuclei were counterstained with Hoechst 33342 (10 μg/mL in H2O at room temperature for 10 min). After washing with 1X DPBS twice, the slides with cells were wet mounted. Cells were imaged using a Zeiss LSM880-Airyscan FAST super-resolution single-point, laser scanning confocal microscope. Images were processed using Zeiss Zen software.
9
Knee Joint Immunofluorescence Staining
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The knee joint sections were incubated with CD31, F4/80, CD206, and iNOS antibodies (1:300) at 4 °C overnight, respectively. Afterwards, samples were incubated with secondary Alexa Fluor 488 antibody or Alexa Fluor cy3 (1:8000; Abcam, UK) for 2 h at room temperature. Nuclei were stained with DAPI (Beyotime, China). Images were captured by a Leica fluorescence microscope.
10
Immunohistochemical Analysis of IL-36 Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Frozen heart tissue sections (10 µm) were incubated with primary antibodies against IL-36R, IL-36α, IL-36β, or IgG control antibodies (1:100 dilution; R & D Systems) and a secondary donkey anti-goat Alexa Fluor-488 antibody (1:100 dilution; Abcam) at room temperature. Sections were also incubated with PE anti-mouse CD31 antibody (1:100 dilution, Biolegend), Alexa Fluor-647 anti-mouse CD106/VCAM-1 antibody (1:100 dilution, Biolegend), or an anti-DNA/RNA damage antibody for oxidative damage detection (1:100 dilution, Abcam). Images were captured using an EVOS FL microscope (ThermoFisher Scientific), and ImageJ was used to quantify mean fluorescence intensity (MFI) for each image.
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!