Cells were seeded at a density of 10,000 cells/cm2 on glass coverslips pretreated with 30 μg/mL poly-L lysine to promote adherence. At the end of the treatment cells were fixed with 4% paraformaldehyde (Euroclone) for 10 min at 4 °C and permeabilized for 10 min at RT with 0.1% (v/v) Triton X-100 (Bio-Rad). After washing, cells were incubated with anti-tau (D1M9X) anti-pH2AX or anti-β-Tubulin (all from Cell Signaling Technology), for additional 45 min. After washing with PBS, cells were incubated for 30 min at RT with Alexa Fluor 594/488-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA, USA). Controls were performed by omitting the primary antibody. Slides were mounted with ProLong Gold antifade mounting medium with DAPI (Cell Signaling Technology). Finally, cells were observed with Zeiss Axio Vert. A1 fluorescence microscope (Zeiss, Jena, Germany) and acquired images were digitally elaborated with a modular image-processing and analysis software (Zen 2012 SP2 Blue Edition).
Alexa fluor 488 594 conjugated secondary antibodies
Manufactured by Jackson ImmunoResearch
Sourced in United States
Alexa Fluor 488/594-conjugated secondary antibodies are fluorescently-labeled antibodies that can be used to detect and visualize target proteins in various applications, such as immunofluorescence, flow cytometry, and western blotting. The Alexa Fluor dyes provide bright and photostable fluorescent signals that can be detected using standard filter sets.
Automatically generated - may contain errors
Lab products found in correlation
Axio vert a1 fluorescence microscope, by Zeiss (2 mentions)
Triton x 100, by Bio-Rad (2 mentions)
Prolong gold antifade mounting medium with dapi, by Cell Signaling Technology (2 mentions)
Fluoview fv10i, by Olympus (2 mentions)
Anti β tubulin, by Cell Signaling Technology (1 mentions)
Cryostat, by Thermo Fisher Scientific (1 mentions)
Ds ri1 camera, by Nikon (1 mentions)
Kim 1, by R&D Systems (1 mentions)
Paraformaldehyde (pfa), by Euroclone (1 mentions)
Eclipse e600, by Nikon (1 mentions)
Mab353, by Merck Group (1 mentions)
Ab15580, by Abcam (1 mentions)
Ab72139, by Abcam (1 mentions)
Ab97959, by Abcam (1 mentions)
Mab5384, by Merck Group (1 mentions)
Image pro, by Media Cybernetics (1 mentions)
Optimal cutting temperature compound, by Sakura Finetek (1 mentions)
Glial fibrillary acidic protein (gfap), by Abcam (1 mentions)
Iba 1, by Abcam (1 mentions)
Gasdermin d, by Santa Cruz Biotechnology (1 mentions)
10 protocols using alexa fluor 488 594 conjugated secondary antibodies
1
Immunocytochemical Evaluation of pH2AX and Tau
2
Kidney Tissue Immunofluorescence Staining
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For immunofluorescence staining, the PLP-fixed and O.C.T.-embedded kidney tissues were sectioned by a cryostat (Thermo Scientific, Cheshire, UK) to 4 μm thick. Then, the cryosections were blocked with 2% BSA in PBS for 30 min and incubated with the following primary antibodies overnight at 4 °C: mouse monoclonal Wnt4 (1:200, Santa Cruz Biotech, Delaware Avenue, CA, USA), goat polyclonal Kim-1 (1:400, R&D Systems, Minneapolis, MN, USA), rabbit monoclonal Ki67 (1:400, Cell Signaling Technology, Danvers, MA, USA, a marker for cell proliferation or regeneration), rabbit polyclonal AQP-1 (1:200, Proteintech, Rosemont, IL, USA, a marker of proximal tubules), goat polyclonal NCCT (1:100, Santa Cruz Biotech, Delaware Avenue, CA, USA, a marker of distal tubules) and rabbit polyclonal NCCT (1:200, Merck, Darmstadt, Germany). Double staining was performed on the same tissue section. On the following day, the sections were washed several times and incubated at room temperature for 1 h with Alexa Fluor 594/488-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA) at dilutions of 1:200. Nuclei were stained using 4, 6-diamidino-2-phenylindole (DAPI). Images were captured using a Nikon DS Ri1 camera (Tokyo, Japan) and processed and analyzed by Image-Pro Plus software.
3
Immunofluorescence Staining of Tau and Tubulin
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Cells were seeded at a density of 10,000 cells/cm2 on glass coverslips pretreated with 30 μg/mL poly-L lysine to promote adherence. At the end of the treatment cells were fixed with 4% paraformaldehyde (Euroclone) for 10 min at 4 °C and permeabilized for 10 min at RT with 0.1% (v/v) Triton X-100 (Bio-Rad). After washing, cells were incubated, according to dilution suggested by manufacturer, with anti-Tau (D1M9X) anti-Phospho-Tau (Thr231) or anti-β-Tubulin (D3U1W) (all from Cell Signaling Technology), for additional 45 min. After washing with PBS, cells were incubated for 30 min at RT with Alexa Fluor 594/488-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA, USA). Controls were performed by omitting the primary antibody. Slides were mounted with ProLong Gold antifade mounting medium with DAPI (Cell Signaling Technology). Finally, cells were observed with Zeiss Axio Vert. A1 fluorescence microscope (Zeiss, Jena, Germany) and acquired images were digitally elaborated with a modular image-processing and analysis software (Zen 2012 SP2 Blue Edition).
4
Immunocytochemistry for Neural Cell Markers
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For immunocytochemistry, cells were fixed in 4% paraformaldehyde at room temperature for 30 min. Then, incubate with 0.3% TritonX-100 (in PBS) for 15 min at room temperature and wash with PBS. The primary antibody is diluted to a suitable concentration with a blocking solution (10% horse serum in PBS). The following primary antibodies were used: anti-A2B5 (MA1-90445, Thermo Fisher Scientific), anti-CNPase (ab44289, Abcam), anti-Ki67 (ab15580, Abcam), anti-Nestin (MAB353, Milipore), anti-NG2 (MAB5384, Milipore), anti-O4 (NL1326V, R&D), anti-RIP (ab72139, Abcam), and anti-SOX2 (ab97959, Abcam). Primary antibodies were washed out with PBS three times after overnight incubation at 4°C. The corresponding Alexa Fluor 488/594 conjugated secondary antibodies (both from Jackson ImmunoResearch Laboratories) were used for 2 h at room temperature. After wash, the ProLongTM Gold Antifade Mountant with DAPI buffer (P36931, Thermo Fisher Scientific) were used for mounting slides. Results were visualized by fluorescent microscopy (Nikon Eclipse E600; Nikon, Melville, NY, United States). Quantitative image analysis was performed using ImagePro (Media Cybernetics).
5
Fluorescent Immunostaining Protocol for Cells and Tissues
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Cell slides were fixed with 4% paraformaldehyde for 15 minutes after treatment. Paraffin-embedded tissue slides were routinely deparaffinized and hydrated and then immersed in citric acid/sodium citrate buffer for 7 minutes at high temperature and high pressure. Cell and tissue slides were blocked with 10% donkey serum in 0.3% Triton X-100 for 60 minutes at room temperature. Slides were incubated in diluent primary antibody overnight at 4°C, washed with PBS 3 times, and incubated in diluent containing Alexa Fluor 488/594-conjugated secondary antibodies (Jackson ImmunoResearch, West Grove, PA, USA) for 1 hour at room temperature. After DAPI staining for 5 minutes and another 3 washes with PBS, the slides were covered in AntiFade Mounting Medium (MCE) and then observed under a fluorescence microscope (Olympus BX53) or a confocal microscope (C2; Nikon, Tokyo, Japan). The primary antibodies used are listed in Supplementary Table S1 .
6
Immunofluorescence Staining of Neuronal Cells
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Immunofluorescence staining was performed as previously described. Briefly, neuronal coverslips were fixed with 4% PFA for 10 min and then permeabilized with blocking buffer comprising 5% goat serum, 1% bovine serum albumin, and 0.3% Triton X-100 at room temperature for 1 h. The samples were incubated overnight with primary antibodies against GSDMD (1:200, Santa Cruz Biotechnology, USA) and microtubule-associated protein-2 (MAP2) (1:500, Millipore Sigma, USA) at 4 °C, then incubated with the appropriate Alexa Fluor-488/594-conjugated secondary antibodies (Jackson ImmunoResearch, USA) and DAPI. Images were obtained through confocal microscopy (Fluoview FV 10i, Olympus, USA) and analyzed in FV10-ASW 2.1 Viewer software.
7
Immunofluorescence Staining Protocol for Cells and Tissue Sections
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Cell slides were fixed with 4% paraformaldehyde (PFA) for 7 minutes after treatment and blocked with 5% donkey serum in 0.1% Triton X-100 for 30 minutes at room temperature. The slides were incubated with the appropriate primary antibodies (VE-cadherin or LECT2 antibodies) at 4°C for at least 16 hours, washed extensively with PBS three times, and incubated with Alexa Fluor 488/594-conjugated secondary antibodies (Jackson ImmunoResearch, West Grove, PA, USA) for 2 hours at room temperature. The slides were then counterstained with 4′,6-diamidino-2-phenylindole (DAPI), washed, and mounted for observation under a microscope (BX53; Olympus, Tokyo, Japan). The primary and secondary antibodies used for immunofluorescence are listed in Supplementary Table S2 . Tissue sections (3 µm) were washed with PBS three times and incubated with 5% donkey serum in 0.3% Triton X-100 at room temperature for 1 hour.27 (link) The subsequent steps were the same as those used for the cell slides. The primary and secondary antibodies used for immunofluorescence are listed in Supplementary Table S2 .
8
Immunofluorescence Analysis of Neuroinflammatory Markers
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Anesthetized mice were successively perfused intracardially with 0.9% sodium chloride and 4% paraformaldehyde (PFA). Mouse brains were soaked in 4% PFA for 12 h and then dehydrated by sucrose solutions with an ascending concentration gradient of 10, 20, and 30% at 4 °C. After fixation in optimal cutting temperature compound (Sakura Finetek, USA), the brains were sliced into 15-μm sections. Brain sections and neuron coverslips were fixed with 4% PFA for 20 min and then permeabilized with blocking buffer comprising 5% goat serum, 1% bovine serum albumin (BSA) and 0.3% Triton X-100 at room temperature for 1 h. The samples were incubated overnight with primary antibodies against LDLR (1:200, Santa Cruz Biotechnology, USA), NeuN (1:500, Abcam, UK), GFAP (1:500, Abcam, UK), Iba-1 (1:100, Abcam, UK), NLRP3 (1:200, Abcam, UK), ASC (1:200, Santa Cruz Biotechnology, USA), Caspase-1 (1:200, Santa Cruz Biotechnology, USA), and Gasdermin D (1:200, Santa Cruz Biotechnology, USA) at 4 °C, followed by incubation with appropriate Alexa Fluor-488/594-conjugated secondary antibodies (Jackson ImmunoResearch) and DAPI. Immunofluorescence images were captured with a microscope (Olympus MX51, Japan). The positive signals were analyzed using ImageJ software.
9
Immunofluorescence Staining of Neurons
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Immuno uorescence staining was performed as previously described. Brie y, neuronal coverslips were xed with 4% PFA for 10 min and then permeabilized with blocking buffer comprising 5% goat serum, 1% bovine serum albumin (BSA) and 0.3% Triton X-100 at room temperature for 1 h. The samples were incubated overnight with primary antibodies against GSDMD (1:200, Santa Cruz Biotechnology, USA) and microtubule associated protein-2 (MAP2) (1:500, MilliporeSigma, USA) at 4 °C, followed by incubation with appropriate Alexa Fluor-488/594-conjugated secondary antibodies (Jackson ImmunoResearch, USA) and DAPI. Images were obtained with confocal microscopy (Fluoview FV 10i, Olympus, USA) and analyzed using FV10-ASW 2.1 Viewer software.
10
Western Blot and Immunofluorescence Assay
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For western blot, cells were harvested after 24 h post transfection or 2 h post treatment with chemicals and lysed with RIPA buffer for 30 min. Cell extraction were separated by SDS-PAGE electrophoresis and analyzed by western blot with corresponding antibodies. The following primary antibodies used in this study were anti-GAPDH (EM1101, 1:1000) and anti-RPA32 (ET7109-41, 1:1000) from HuaBio; anti-γH2AX (#05-636, 1:1000) and anti-H2AX (#07-627, 1:1000) from Millipore; anti-p-CHK1 (#2348, 1:1000), anti-p-CHK2 (#2661, 1:1000), anti-p-P53 (#9284, 1:1000), anti-CHK2 (#2662, 1:1000) and anti-P53 (#2524, 1:1000) from Cell Signaling Technology; anti-CHK1 (sc-8408, 1:1000) from Santa Cruz; anti-p-RPA32 (ab87277, 1:1000) and anti-53BP1 (ab21083, 1:500) from Abcam; anti-BRCA1 antibody was a gift from the Lu Lab.
For immuno uorescence staining, cells were grown on glass coverslips in a 24-well plate and xed with 4% paraformaldehyde after culturing for 24 h with or without chemical treatment. Cells were permeabilized with 0.1% Triton X-100, followed by blocking in 5% bovine serum albumin in PBS. Cells were probed with primary antibodies and Alexa Fluor 488/594-conjugated secondary antibodies (Jackson ImmunoResearch), stained with DAPI and imaged by a uorescence microscope (Leica DM4000).
For immuno uorescence staining, cells were grown on glass coverslips in a 24-well plate and xed with 4% paraformaldehyde after culturing for 24 h with or without chemical treatment. Cells were permeabilized with 0.1% Triton X-100, followed by blocking in 5% bovine serum albumin in PBS. Cells were probed with primary antibodies and Alexa Fluor 488/594-conjugated secondary antibodies (Jackson ImmunoResearch), stained with DAPI and imaged by a uorescence microscope (Leica DM4000).
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