The cells were transferred into immunofluorescence plates (SB‐Shifix20; Shikhar Biotech, Lalitpur, Nepal); washed with PBS three times, 5 minutes each; permeabilized using 0.3% Triton X‐100 for a total of 15 minutes; and blocked with 1% FBS at room temperature for 1 hour. The samples were incubated at room temperature overnight with mouse monoclonal anti‐βIII‐tubulin, anti‐F4/80 or rabbit polyclonal anti‐iNOS or anti‐NeuN (both 1:500, Abcam, Cambridge, MA). After the primary antibody was removed, the samples were washed with PBS three times, 5 minutes each, followed by incubation in the dark at room temperature for 40 minues with either Alexa Fluor 594‐conjugated donkey anti‐mouse secondary antibody or Alexa Fluor 488‐conjugated donkey anti‐rabbit secondary antibody (both 1:1500, Abcam).
Alexa fluor 488 conjugated donkey anti rabbit secondary antibody
Manufactured by Abcam
Alexa Fluor 488-conjugated donkey anti-rabbit secondary antibody is a detection reagent used to identify and visualize rabbit primary antibodies in various applications such as immunofluorescence and Western blotting. The antibody is conjugated to the fluorescent dye Alexa Fluor 488, which has an excitation maximum at 495 nm and an emission maximum at 519 nm, allowing for green fluorescent detection.
Automatically generated - may contain errors
Lab products found in correlation
Growth supplement, by Lonza (1 mentions)
Poly l lysine (pll), by Merck Group (1 mentions)
Nhlfs, by Lonza (1 mentions)
Fibroblast basal medium, by Lonza (1 mentions)
Alpha tubulin antibody, by Santa Cruz Biotechnology (1 mentions)
Sb431542, by R&D Systems (1 mentions)
Zen black edition software, by Zeiss (1 mentions)
Lc3a b antibody, by Cell Signaling Technology (1 mentions)
Fluorescence microscope, by Nikon (1 mentions)
Cy3 goat anti rabbit secondary antibody, by Abcam (1 mentions)
Donkey serum, by Solarbio (1 mentions)
Dm2500, by Leica (1 mentions)
Alexa fluor 647 conjugated donkey anti rabbit secondary antibody, by Abcam (1 mentions)
Frozen slicer, by Leica (1 mentions)
Anti gaba, by Merck Group (1 mentions)
Ab150075, by Abcam (1 mentions)
Anti c fos, by Cell Signaling Technology (1 mentions)
Anti glutamate, by Merck Group (1 mentions)
Dm1860, by Leica (1 mentions)
Ab150073, by Abcam (1 mentions)
5 protocols using alexa fluor 488 conjugated donkey anti rabbit secondary antibody
1
Immunofluorescence Staining of Neural and Immune Markers
2
Fibroblast Characterization and Activation
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Fibroblast basal medium, growth supplements, and NHLFs were purchased from Lonza (Walkersville, MD). Human-derived transforming growth factor beta (TGFβ), as well as the TGFβ receptor blocker SB431542, was obtained from R&D Systems, Inc. (Minneapolis, MN). Poly-L-lysine was purchased from Sigma-Aldrich (St. Louis, MO). The anti-Collagen I antibody was purchased from Fitzgerald (Acton, MA), while the alpha tubulin antibody was purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX). Alexa-fluor 488-conjugated donkey anti-rabbit secondary antibody, alexa-fluor 488-conjugated alpha-smooth muscle actin (α-SMA) primary antibody, and the beta actin primary antibody were purchased from Abcam (Cambridge, MA). ProLong gold antifade with DAPI mountant was obtained from Molecular Probes (Thermo Fisher Scientific, Waltham, MA).
3
Visualizing Mitochondria and Autophagosomes
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Cells seeded on 8-well microscopy
slides were fixed with 2% glutaraldehyde solution (cat. no. 340855,
Sigma Aldrich) by incubating at 4 °C for 15 min. 0.1 M glycine
solution was applied to quench residual aldehydes for 1 h at room
temperature that was followed by permeabilization with 0.25% Triton
X-100 in PBS at room temperature for 15 min. Prior to antibody incubation,
cells were blocked with 3% Blocker BSA in PBS (cat. no. 37525, Thermo
Fisher Scientific) by incubating for 1 h at room temperature. Mitochondria
were labeled by incubating the cells at 4 °C overnight with an
Alexa Fluor 488-conjugated anti-mitochondria antibodies (cat. no.
MAB1273A4, Merck Millipore) in blocking solution at 1:500 dilution.
For imaging autophagosome formation, slides were incubated overnight
with LC3A/B antibodies (Cell Signaling Technologies, polyclonal) at
1:200 dilution followed by incubating with an Alexa Fluor 488-conjugated
donkey anti-rabbit secondary antibody (Abcam, polyclonal) for an hour
at room temperature. Slides were mounted with Antifading Mounting
Medium with DAPI (cat. no. SCR-038448, Dianova) and visualized under
a Zeiss LSM 880 Airyscan inverted microscope at 63× magnification
with immersion oil. Images were obtained using Zeiss Zen software
(Black Edition).
slides were fixed with 2% glutaraldehyde solution (cat. no. 340855,
Sigma Aldrich) by incubating at 4 °C for 15 min. 0.1 M glycine
solution was applied to quench residual aldehydes for 1 h at room
temperature that was followed by permeabilization with 0.25% Triton
X-100 in PBS at room temperature for 15 min. Prior to antibody incubation,
cells were blocked with 3% Blocker BSA in PBS (cat. no. 37525, Thermo
Fisher Scientific) by incubating for 1 h at room temperature. Mitochondria
were labeled by incubating the cells at 4 °C overnight with an
Alexa Fluor 488-conjugated anti-mitochondria antibodies (cat. no.
MAB1273A4, Merck Millipore) in blocking solution at 1:500 dilution.
For imaging autophagosome formation, slides were incubated overnight
with LC3A/B antibodies (Cell Signaling Technologies, polyclonal) at
1:200 dilution followed by incubating with an Alexa Fluor 488-conjugated
donkey anti-rabbit secondary antibody (Abcam, polyclonal) for an hour
at room temperature. Slides were mounted with Antifading Mounting
Medium with DAPI (cat. no. SCR-038448, Dianova) and visualized under
a Zeiss LSM 880 Airyscan inverted microscope at 63× magnification
with immersion oil. Images were obtained using Zeiss Zen software
(Black Edition).
4
Immunohistochemical Analysis of M2 Microglia and NLRP3 Inflammasome
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Briefly, brain sections were incubated in citrate buffer at 60°C for 30 minutes and in 5% goat serum at room temperature for 1 hour. The sections were then incubated with rabbit anti-CD206 primary antibody (a marker for M2 microglia; 1:500, Abcam, Cat# ab64693, RRID: AB_1523910) at 4°C overnight and Cy3 goat anti-rabbit secondary antibody (1:500, Abcam, Cat# ab6939, RRID: AB_955021) at room temperature for 1 hour. Finally, anti-fluorescence quenching sealing tablets containing 4,6-diamidino-2-phenylindole were used to stain the nuclei, and fluorescent images were taken using a fluorescence microscope (Nikon, Tokyo, Japan).
BV2 cells were treated with the drugs for 24 hours, and then were fixed with 4% paraformaldehyde for 20 minutes, followed by permeabilization and blocking with 0.1% Triton X-100 and 5% donkey serum (Solarbio) at room temperature for 1 hour. The cells were then incubated with rabbit anti-NLRP3 antibody (1:500, Abcam, Cat# ab214185, RRID: AB_2819003) at 4°C overnight followed by incubation with Alexa Fluor 488-conjugated donkey anti-rabbit secondary antibody (1:500, Abcam, Cat# ab150073, RRID: AB_2636877) at room temperature for 2 hours. Finally, 4,6-diamidino-2-phenylindole was used to stain the nuclei, and fluorescent images were taken. The level of NLRP3 was observed using a fluorescence microscope.
BV2 cells were treated with the drugs for 24 hours, and then were fixed with 4% paraformaldehyde for 20 minutes, followed by permeabilization and blocking with 0.1% Triton X-100 and 5% donkey serum (Solarbio) at room temperature for 1 hour. The cells were then incubated with rabbit anti-NLRP3 antibody (1:500, Abcam, Cat# ab214185, RRID: AB_2819003) at 4°C overnight followed by incubation with Alexa Fluor 488-conjugated donkey anti-rabbit secondary antibody (1:500, Abcam, Cat# ab150073, RRID: AB_2636877) at room temperature for 2 hours. Finally, 4,6-diamidino-2-phenylindole was used to stain the nuclei, and fluorescent images were taken. The level of NLRP3 was observed using a fluorescence microscope.
5
Neuronal Activity Mapping in Brain Sections
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Animals were placed in the behavioral field for 2 h on day 21 and were deeply anaesthetized with xylazine (10 mg/kg) and ketamine (100 mg/kg) for transcardial perfusion with saline followed by 4% paraformaldehyde (0.1 M PB pH 4.5). Brains were extracted and post-fixed in 4% paraformaldehyde overnight at 4°C and immersed in 30% sucrose in 0.01 M PBS (pH 7.4) for cryoprotection. Coronal sections (40 μm) were obtained with a Frozen Slicer (Leica DM1860). Sections were rinsed in PBS three times for 10 min each time, permeabilized in 0.3% TritonX-100 for 30 min at 37°C, blocked for 1 h with 5% normal donkey serum at room temperature and then incubated with primary antibodies, including anti-c-Fos (1:500, rabbit, Cell Signaling Technology), anti-GABA (1:500, rabbit, Sigma Aldrich), anti-glutamate (1:500, rabbit, Sigma Aldrich) and anti-somatostatin (1:500, rabbit, ab108456), at 4°C for 24 h. Sections were then rinsed in PBS three times for 10 min each time, and incubated with Alexa Fluor 488-conjugated donkey anti-rabbit secondary antibody (1:500, abcam, ab150073) or Alexa Fluor 647-conjugated donkey anti-rabbit secondary antibody (1:500, abcam, ab150075) for 1.5 h at room temperature. Fluorescence staining images were observed under fluorescence microscopy (Leica DM2500).
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