Cells were seeded into six-well plates. After treatment, the cells were fixed with 4% PFA for 20 min. Then, the cells were permeabilized for 5 min in PBS with 0.25% Triton X-100 (T8787; Sigma-Aldrich). After blocking with 5% goat serum for 30 min, the cells were incubated overnight at 4°C with a primary antibody in a humidified box. The tissues were treated with a secondary antibody (Alexa Fluor 488–labeled donkey anti-rabbit antibody; Invitrogen) for 1 h, after washing for three times with 0.1% TBST. Finally, DAPI (P0131; Beyotime) was added to the cells to stain the cellular nucleus.
Alexa fluor 488 labeled donkey anti rabbit antibody
Manufactured by Thermo Fisher Scientific
Sourced in United States
Alexa Fluor 488-labeled donkey anti-rabbit antibody is a secondary antibody used in immunoassays. It binds to primary rabbit antibodies and is labeled with the Alexa Fluor 488 fluorescent dye for detection purposes.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Beyotime (1 mentions)
Alexa fluor plus 555, by Thermo Fisher Scientific (1 mentions)
Gfp polyclonal antibody, by Thermo Fisher Scientific (1 mentions)
Active caspase 3, by Cell Signaling Technology (1 mentions)
α actin, by Abcam (1 mentions)
Mmp 8, by Abcam (1 mentions)
Tnf α, by Abcam (1 mentions)
6 diamino 2 phenylindole dapi, by Beyotime (1 mentions)
Sirius red, by Merck Group (1 mentions)
β actin, by Abcam (1 mentions)
Atorvastatin, by Pfizer (1 mentions)
Proteinase k solution, by Merck Group (1 mentions)
Antifade reagent containing dapi, by Thermo Fisher Scientific (1 mentions)
4 protocols using alexa fluor 488 labeled donkey anti rabbit antibody
1
Immunofluorescence Staining of Cells
2
Immunostaining of Cochlear Tissues
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Cochlear explants or zebrafish larvae were fixed with 4% paraformaldehyde for 1 h, washed three times with PBS, and permeabilized with 1% Triton X-100 in PBS at 25°C for 10 min. Permeabilized samples were then blocked with 10% bovine serum albumin in PBS for 1 h, followed by incubation with the primary antibodies overnight at 4°C. The primary antibodies used in this experiment were myosin VIIa rabbit polyclonal antibody (25–6790; Proteus Biosciences) and GFP polyclonal antibody (A11122; Invitrogen). The samples were rinsed three times with PBS and incubated with the relevant secondary antibodies at 37°C for 1 h away from light. The secondary antibodies were goat antirabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 555 (A32732; Invitrogen), and Alexa Fluor 488-labeled donkey antirabbit antibody (A21206; Invitrogen). The nuclei were labeled with DAPI (D523; Dojindo Laboratories) for 10 min at room temperature, and the fluorescent signals were captured under a fluorescence microscope.
3
Molecular Mechanisms of XZK-mediated Cardioprotection
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XZK powder was kindly provided by the WBL Peking University Biotech Co., Luye Pharma Group (Beijing, China). 7-ketocholesterol (7-KC), Oil red O and sirius red were purchased from Sigma-Aldrich (St. Louis, MO, USA). Atorvastatin was obtained from Pfizer Ltd. (New York, NY, USA). The antibodies against α-actin, β-actin, MMP8, MMP13, TNFα, phosphorylated IRE1α (p-IRE1α), IRE1α, eIF2α, PERK and IκBα were from Abcam (Cambridge, MA, USA); antibodies against BiP, phosphorylated PERK (p-PERK), phosphorylated eukaryotic initiation factor 2α (p-eIF2α), spliced x-box binding protein 1 (s-XBP1), cleaved PARP, and active caspase-3 were from Cell Signaling Technology (Beverly, MA, USA); and antibodies against CCAAT-enhancer-binding protein homologous protein (CHOP) and ATF6 were from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). Secondary antibodies including Alexa Fluor 488-labeled donkey anti-rabbit antibody, Alexa Fluor 647-labeled goat anti-rat antibody, and Alexa Fluor 555-labeled donkey anti-mouse antibody were from Invitrogen (Carlsbad, CA, USA). The TUNEL assay kit (In Situ Cell Death Detection kit) was from Roche (Mannheim, Germany), real-time PCR (qPCR) reagent kits were from TAKARA Biotechnology (Dalian, China), and 6-diamino-2-phenylindole (DAPI) was from Beyotime Biotechnology (Shanghai, China).
4
Immunofluorescence Staining of Lung Tissue
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Paraffin sections were de-waxed in Histo-Clear solutions twice (National Diagnostics) and rehydrated first in absolute ethanol three times, and then once each in 90% ethanol, 70% ethanol and 50% ethanol. H&E staining was processed according to standard protocol. For immunofluorescence staining, antigen retrieval was performed by incubating the lung sections with proteinase K solution (Sigma-Aldrich, 20 mg/ml proteinase K in 50 mM Tris-Cl, 1 mM EDTA, pH 8.0) at 37°C for 30 min. Sections were then blocked in blocking buffer (3% BSA, 0.2% Triton X-100 in PBS) for 1 hr. Polyclonal rabbit anti-CCSP (also known as SCGB1A1) antibody (US Biological, catalogue number C5828) was used at 1:100 dilution. Goat anti-pro-SPC antibody (Santa Cruz Biotechnology, catalogue number sc-7706) and goat anti-Podopanin antibody (R&D Systems, catalogue number AF3244) were used at 1:100 dilution. Incubation was performed at 4°C overnight in blocking buffer. Secondary Alexa Fluor 488-labeled donkey anti-rabbit antibody (Invitrogen, catalogue number A21206) and Alexa Fluor 546-labeled donkey anti-goat antibody (Invitrogen, catalogue number A11056) were used at 1:100 dilution. Incubation was performed at room temperature for 1 hr. Cover slips were mounted on stained sections with antifade reagent containing DAPI (Invitrogen).
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