4 μm paraformaldehyde-fixed paraffin-embedded tissue sections were deparaffinized in xylene and rehydrated followed by antigen retrieval in 10 mM sodium citrate and a blocking step with 10% normal donkey serum/5% BSA/PBS. Chicken anti-GFP (Abcam), rabbit anti-Salmonella O4 antigen (Abcam), rat anti-LAMP1 (Developmental Studies Hybridoma Bank), rabbit anti-cleaved caspase-3 (Cell Signaling), rabbit anti-cleaved caspase-8 (Cell Signaling), rat anti-PMN (Ly6-6B2, SeroTec) and mouse anti-E-cadherin (BD Transduction Laboratories) antibodies as well as the indicated fluorophore-conjugated secondary antibodies (Jackson ImmunoResearch) were used. Fluorescein-conjugated (Vector) or AF647-conjugated (Invitrogen) Wheat Germ Agglutinin (WGA) was used to detect the mucus layer. Slides were subsequently mounted in DAPI mounting medium (Vector) and images were taken using a Zeiss ApoTome.2 system microscope connected to a Axiocam 506 digital camera. Images were formatted using the ZEN 2.3 imaging software.
Rabbit anti cleaved caspase 8
Manufactured by Cell Signaling Technology
Sourced in United States
Rabbit anti-cleaved caspase-8 is a primary antibody produced in rabbits that specifically recognizes the cleaved form of caspase-8. Caspase-8 is a key enzyme involved in the initiation of apoptosis, or programmed cell death.
Automatically generated - may contain errors
Lab products found in correlation
Rabbit anti cleaved caspase 3, by Cell Signaling Technology (4 mentions)
Rabbit anti cleaved caspase 9, by Cell Signaling Technology (3 mentions)
Fluorophore conjugated secondary antibody, by Jackson ImmunoResearch (1 mentions)
Mouse anti e cadherin, by BD (1 mentions)
Dapi mounting medium, by Vector Laboratories (1 mentions)
Chicken anti gfp, by Abcam (1 mentions)
Human tnf α, by Thermo Fisher Scientific (1 mentions)
Gsk 872, by Merck Group (1 mentions)
Mouse anti gfp, by Thermo Fisher Scientific (1 mentions)
Mouse anti v5, by Thermo Fisher Scientific (1 mentions)
Rabbit anti myc, by Cell Signaling Technology (1 mentions)
Rabbit anti ha, by Cell Signaling Technology (1 mentions)
Cycloheximide (chx), by Merck Group (1 mentions)
Z vad fmk, by Merck Group (1 mentions)
Necrostatin 1, by Merck Group (1 mentions)
Mouse anti flag hrp, by Merck Group (1 mentions)
Mouse anti lmp1, by Agilent Technologies (1 mentions)
Mouse anti β actin, by Santa Cruz Biotechnology (1 mentions)
Goat anti rabbit igg hrp, by Santa Cruz Biotechnology (1 mentions)
Goat anti mouse igg hrp, by Santa Cruz Biotechnology (1 mentions)
5 protocols using rabbit anti cleaved caspase 8
1
Multicolor Immunofluorescence Staining of FFPE Tissue
2
Necroptosis Signaling Pathway Reagents
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Human TNFα and Smac mimetic (GDC-0152) were purchased from Peprotech (Rocky Hill, NJ, USA) and Selleck (Houston, TX, USA), respectively. Necrostatin-1, z-VAD-fmk and CHX were obtained from Sigma-Aldrich (Saint Louis, MO, USA). GSK’872 was from Merck-Millipore (Billerica, MA, USA). Antibodies used were rabbit anti-RIPK1, rabbit anti-Myc, rabbit anti-HA, mouse anti-Myc, rabbit anti-cleaved caspase-3, and rabbit anti-cleaved caspase-8 from Cell Signaling Technology (Danvers, MA, USA), rabbit anti-TRAF2, mouse anti-β-actin, goat anti-rabbit IgG-HRP, and goat anti-mouse IgG-HRP from Santa Cruz Biotechnology (Santa Cruz, CA, USA), rabbit anti-RIPK3, rabbit anti-phosphorylated RIPK3, rabbit anti-phosphorylated MLKL, and mouse anti-phosphorylated IκBα from Abcam (Cambridge, MA, USA), rabbit anti-MLKL, mouse anti-Flag, and mouse anti-Flag-HRP from Sigma-Aldrich, mouse anti-RIPK1 from BD Biosciences (San Jose, CA, USA), mouse anti-LMP1 from DAKO (Glostrup, Denmark), mouse anti-V5 from Invitrogen (Carlsbad, CA, USA), mouse anti-GFP from Thermo Fisher (Waltham, MA, USA).
3
Glutamate-Induced Apoptosis Pathways in Astrocytes
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After astrocytes were treated with 10 mM glutamate and different doses of morphine for 24 h, the cells were lysed by RIPA lysis buffer (Beyotime, Haimen, China) according to the protocol. Western blot was carried out as previously described with minor modification [42 (link)]. The primary antibodies used in experiments were: rabbit anti-cleaved caspase-8, rabbit anti-cleaved caspase-9, rabbit anti-cleaved caspase-3, rabbit anti-phosphor-eIF2α, rabbit anti-eIF2α, rabbit anti-ATF4, mouse anti-CHOP, rabbit anti-IRE1α, rabbit anti-XBP-1, rabbit anti-phosphor-JNK and rabbit anti-GAPDH antibodies (Cell Signaling Technology, Beverly, MA, USA), mouse anti-β-actin antibody (Sigma-Aldrich, St. Louis, MO, USA). Secondary antibodies were purchased from Jackson Laboratory (Sacramento, CA, USA). Each blot was repeated at least three times, the optical density of each band was measured by Image J software (National Institutes of Health, Bethesda, MD, USA).
4
Western Blot Analysis of Apoptosis and Autophagy
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RIPA lysis buffer was used to extract proteins from brain tissues, and then the bicinchoninic acid protein assay kit (Beyotime, China) was used to determine the protein concentration. Equal amounts of protein were subjected to 10% SDS-PAGE prior to transfer onto PVDF membranes (Millipore). After being blocked with 5% nonfat milk, the membranes were then incubated with proper antibodies, including anti-rabbit Bcl-2 (Abcam, 1:1000), anti-rabbit Bax (Abcam, 1:1000), anti-rabbit cleaved Caspase-9 (Cell Signaling, 1:1000), anti-rabbit cleaved Caspase-8 (Cell Signaling, 1:1000), anti-rabbit cleaved Caspase-3 (Abcam, 1:500), anti-rabbit LC3 (Abcam, 1:3000), anti-rabbit p62 (Proteintech, 1:1000), anti-rabbit Beclin1 (Abcam, 1:1000), anti-rabbit ASK1 (Abcam, 1:500), rabbit anti-phospho-ASK1 (Thermo Fisher, 1:500), anti-mouse JNK (Proteintech, 1:3000), rabbit anti-phospho-JNK (Abcam, 1:1000), and anti-mouse β-actin (Proteintech, 1:2000). The mouse β-actin was used as internal reference protein. After being washed, membranes were then incubated with anti-rabbit IgG (H+L) (CST, 1:15000) or anti-mouse IgG (CST, 1:15000) fluorescent secondary antibodies. Densiometric scan and Image J were used to quantify the relative protein levels.
5
Western Blotting Antibody Validation
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Western blotting was performed as previously described (Jiang et al., 2010 (link); Wang et al., 2014 (link)). Anti-rabbit-IQGAP1 antibody was purchased from Proteintech, USA. Anti-rabbit-His, anti-rabbit-UAP56, anti-rabbit-ARHGAP24, anti-rabbit-Cleaved-Caspase-8, anti-rabbit-Cleaved-Caspase-3, anti-rabbit-Cleaved-Caspase-9, anti-rabbit-p-ERK1/2, anti-rabbit-ERK1/2, and anti-rabbit-smurf1 antibodies were purchased from Cell Signaling Technology, USA. Anti-β-actin antibody was purchased from Sigma-Aldrich, USA.
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