The spinal cord tissues of each group were fixed with 4% paraformaldehyde at 4 °C. The spinal cord tissue was placed at the bottom of 50 mL centrifuge tube and dehydrated in 10%, 20%, and 30% sucrose solution. Twelve micrometers frozen sections were made and detected. All sections were blocked with blocking solution [10% goat serum, 3% bovine serum albumin (BSA), and 0.1% Triton X-100] for 1 h at 37 °C and then incubated overnight at 4 °C with Rabbit-anti-Active-Caspase3 (1:500, ab49822, Abcam), Rabbit-anti-Caspase3 (1:500, ab184787, Abcam), Rabbit-anti-LC3 (1:100, ab128025, Abcam), Rabbit-anti-LAMP2 (1:400, L0668, Sigma), Rabbit-anti-Bcl-2 (1:500, NB100-56101, NOVUS) and Rabbit-anti-Bax (1:500, ab12503, Abcam); 0.01 M phosphate buffer saline (PBS) was used to wash them for 10 min at 3 times and followed by incubating with a mixture of FITC- or Cy3-conjugated secondary antibodies for 2 h at room temperature and then being washed again with PBS for 10 min at 3 times. The stained sections were examined with a Leica fluorescence microscope (Leica DM 5000B, Germany).
L0668
Manufactured by Merck Group
Sourced in United States
The L0668 is a laboratory equipment product from Merck Group. It is designed for general-purpose laboratory applications. The core function of the L0668 is to provide a reliable and efficient tool for laboratory tasks.
Automatically generated - may contain errors
Lab products found in correlation
Ab49822, by Abcam (3 mentions)
Ab128025, by Abcam (3 mentions)
Ab184787, by Abcam (3 mentions)
Dm5000b, by Leica (2 mentions)
Ab12503, by Abcam (2 mentions)
Nb100 56101, by Novus Biologicals (2 mentions)
Ab24170, by Abcam (2 mentions)
Nb400 129, by Novus Biologicals (2 mentions)
Ab292, by Abcam (1 mentions)
α smooth muscle actin α sma clone 1a4, by Merck Group (1 mentions)
Ab128859, by Abcam (1 mentions)
Ab2810, by Abcam (1 mentions)
Ab186734, by Abcam (1 mentions)
Pm045, by Abcam (1 mentions)
Rabbit anti akt, by Cell Signaling Technology (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
A1978, by Merck Group (1 mentions)
Mouse anti β actin, by Merck Group (1 mentions)
Rabbit anti lamp2, by Merck Group (1 mentions)
Rabbit anti p akt, by Cell Signaling Technology (1 mentions)
6 protocols using l0668
1
Immunofluorescent Analysis of Spinal Cord Tissue
2
Immunohistochemical Profiling of Lysosomal Markers
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We used Abs against the C-terminal region of rat and mouse LAMP-1 (ab24170; Abcam, Cambridge, MA), LAMP-2 (L0668; Sigma-Aldrich, St. Louis, MO), LAMP-2a (51-2200; Invitrogen/Life Technologies, Carlsbad, CA), and lysosomal integral membrane protein type-2 (LIMP-2) (NB400-129; Novus Biologicals Oakville, ON, Canada); and against full-length/luminal mouse LAMP-1 (1d4b; Iowa Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA), and mouse LAMP-2 (GL2A7; Iowa Developmental Studies Hybridoma Bank). The following Abs were used for immunofluorescence and immunohistochemical analyses: collagen-1 (ab292; Abcam), Ki67 (IHC-00375; Bethyl Laboratories, Inc. Montgomery, TX), α-smooth muscle actin (α-SMA) (clone 1A4; Sigma-Aldrich), insulin (4590; Cell Signaling Technology, Beverly, MA), CD68 (ABIN181836; Antibodies Online, Atlanta, GA), and CD206 (OASA05048; Aviva Systems Biology, San Diego, CA).
3
Autophagy and Mitochondrial Autophagy Assay
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The authors declare that all supporting data are available within the article and its online supplementary files. An expanded Methods section is available in Data S1 . Autophagy and mitochondrial autophagy were analyzed using electron microscopy, immunohistochemistry, and immunoblots with antibodies against LC3 (light chain 3) (MBL, M186‐3), p62 (SQSTM1; MBL, PM045), Ulk1 (Abcam, ab128859), LAMP2 (lysosome‐associated membrane protein 2; Sigma, L0668), TOMM20 (translocase of outer mitochondrial membrane 20; Abcam, ab186734), and Rab9 (Abcam, ab2810).
4
Protein Expression Profiling of Apoptosis Markers
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The extracted protein was quantified by bicinchoninic acid (BCA) analysis, followed by electrophoresis separation on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). After transferring to a polyvinylidene fluoride (PVDF) membrane (Millipore, Bedford, MA, USA), the membrane was blocked with 5% non-fat dry milk in Tris-buffered saline (TBS, pH 7.4) and incubated with Rabbit-anti-Caspase 3 (1:1,000, ab184787, Abcam, Cambridge, MA, USA), Rabbit-anti-active-Caspase 3 (1:1,000, ab49822, Abcam), Rabbit-anti-Bax (1:1,000, ab12503, Abcam), Rabbit-anti-Bcl2 (1:1,000, NB100-56101, NOVUS), Rabbit-anti-LC3 (1:500, ab128025, Abcam), Rabbit-anti-TNFR1 (1:1000, 21574-1-AP, Proteintech, Rosemont, IL, USA), Rabbit-anti-TNFR2 (1:1,000, 19272-1-AP, Proteintech), Rabbit-anti-p-Akt (1:1,000, 8200S, Cell Signaling Technology, Danvers, MA, USA), Rabbit-anti-Akt (1:1,000, C67E7, Cell Signaling Technology), Mouse-anti-β-actin (1:5,000, A1978, Sigma-Aldrich, St. Louis, MO, USA), and Rabbit-anti-LAMP2 (1:1,500, L0668, Sigma), respectively, at 4 °C overnight. After being washed with TBST (TBS with 0.1% Tween 20) and incubated with an anti-rabbit or anti-mouse horseradish peroxidase-conjugated secondary antibody, the protein was visualized using Beyo ECL Star (Beyotime, Jiangsu, China).
5
Immunofluorescence Analysis of Cell Death Markers
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All cell sections were blocked with blocking solution [10% goat serum, 3% bovine serum albumin (BSA), 0.1% Triton X-100] for 30 min at 37 °C. Then, the sections were incubated with primary antibodies Rabbit-anti-Caspase 3 (1:500, ab184787, Abcam), Rabbit-anti-active-Caspase 3 (1:500, ab49822, Abcam), Rabbit-anti-LC3 (1:100, ab128025, Abcam), Rabbit-anti-TNFR1 (1:400, 21574-1-AP, Proteintech), Rabbit-anti-TNFR2 (1:100, 19272-1-AP, Proteintech), and Rabbit-anti-LAMP2 (1:400, L0668, Sigma), respectively, at 4 °C overnight. After being washed with 0.01 M PBS and incubated with a mixture of FITC- or Cy3-conjugated secondary antibodies for 2 h at room temperature, the stained sections were examined with a Leica fluorescence microscope (Leica DM5000B, Wetzlar, Germany).
6
Immunoblot and Immunofluorescence Antibody Protocol
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The following antibodies were used for immunoblot analysis and immunofluorescence labeling: anti-cathepsin C (dilution: 1:1000, sc-5647, Santa Cruz Biotechnology, RRID: AB_2086961), anti-trypsin (dilution: 1:1000, sc-137077, Santa Cruz Biotechnology, RRID: AB_2300318), anti-amylase (dilution: 1:1000, sc-46657, Santa Cruz Biotechnology, RRID: AB_626668), anti-cathepsin B (dilution: 1:1000, MAB965, R&D systems, RRID: AB_2086935), anti-cathepsin L (dilution: 1:1000, MAB9521, R&D systems, RRID: AB_2087829), anti-α/β-tubulin (dilution: 1:1000, #2148, Cell Signaling, RRID: AB_2288042), anti-Rab7 (dilution: 1:1000 (for Immunoblot) and 1:100 (for immunofluorescence), #9367, Cell Signaling, RRID: AB_1904103), anti-syncollin (dilution: 1:1000, ab178415, Abcam), anti-LAMPI (dilution: 1:1000, ab24170, Abcam, RRID: AB_775978), anti-GAPDH (dilution: 1:1000, H86504M, Meridian Life Science, RRID: AB_151542), anti-LIMPII (dilution: 1:1000, NB400-129, Novus Biologicals, RRID: AB_2301298), anti-LAMPII (dilution: 1:1000, L0668, Sigma Aldrich, RRID: AB_477154), anti-rat (dilution: 1:16,000, HAF005, R&D systems, RRID: AB_1512258), anti-goat (dilution: 1:16,000, sc-2020, Santa Cruz Biotechnology, RRID: AB_631728), anti-rabbit (dilution: 1:16,000 (for immunoblots) or 1:200 (for immunofluorescence), NA934V, Amersham) and anti-mouse (dilution: 1:16,000, NA931V, Amersham).
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