Adult mice were anesthetized with an overdose of pentobarbital and then transcardially perfused with 4% paraformaldehyde (PFA). Mouse brain was dissected and fixed in 4% PFA for 4 h. After cryoprotection in 30% sucrose, brain sections (35 μm) were cut on a cryostat microtome (Leica CM1950, Leica Biosystems, Wetzlar, Germany). After rinsing with PBS and 0.3% Triton-X in 0.1 M PBS (PBST), the brain sections were blocked with 2% (w/v) bovine serum (BSA) in PBST for 1 h. Then, the brain sections were incubated with primary antibodies at 4°C for 48 h and secondary antibodies at room temperature for 2 h. Images were collected using a Zeiss LSM510 Meta or Nikon A1 confocal microscope and analyzed using FIJI. The antibodies used were as follows: anti-choline acetyltransferase (1:200, goat, AB144P, MERCK, Kenilworth, NJ, United States), anti-NK1R (S8305, rabbit, 1:5,000, Sigma-Aldrich, St. Louis, MO, United States), goat anti-rabbit for NK1R (Cy3 and Alexa Fluor 488, Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, United States), donkey anti-goat for choline acetyltransferase (Cy3 and Alexa Fluor 488, Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, United States), and Cy3-streptavidin (1:500).
S8305
Manufactured by Merck Group
Sourced in United States
The S8305 is a laboratory equipment product manufactured by Merck Group. It serves as a general-purpose tool for various laboratory applications. The device's core function is to perform specific tasks or procedures required in a laboratory setting. Further details about the intended use or specific capabilities of the S8305 are not available.
Automatically generated - may contain errors
Lab products found in correlation
Ab16502, by Abcam (2 mentions)
Lsm 510 meta, by Nikon (1 mentions)
Cm1950, by Leica (1 mentions)
Cy3 streptavidin, by Jackson ImmunoResearch (1 mentions)
Alexa fluor 488, by Jackson ImmunoResearch (1 mentions)
Cyanine3 (cy3), by Jackson ImmunoResearch (1 mentions)
Ab144p, by Merck Group (1 mentions)
Ab34712, by Abcam (1 mentions)
Bovine serum albumin (bsa), by Solarbio (1 mentions)
Kit 9720, by Beijing Strong Biotechnologies (1 mentions)
Confocal laser scanning microscope, by Olympus (1 mentions)
Ab16046, by Abcam (1 mentions)
Alexa fluor 488 goat anti mouse igg1, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 594 goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Ab124957, by Abcam (1 mentions)
Ab39012, by Abcam (1 mentions)
Ab2064, by Abcam (1 mentions)
Ab36861, by Abcam (1 mentions)
Enhanced chemiluminescent reagent, by Thermo Fisher Scientific (1 mentions)
Ab76003, by Abcam (1 mentions)
4 protocols using s8305
1
Immunohistochemical Analysis of Cholinergic Neurons
2
Immunohistochemical and Immunofluorescence Analysis of Cartilage and Neurogenic Markers
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The sections were dewaxed to water and then autoclaved to achieve antigen repair. UltraSensitive™ SP kits were used to measure protein levels in samples (KIT-9720, MXB biotechnologies, China). The endogenous enzyme was inactivated with 3% H2O2, then the sections were closed with 5% BSA (Beijing Solarbio Science & Technology Co., Ltd.). Col II primary antibody (ab34712, AbCam, USA, 1:200) was incubated overnight and biotin-labeled IgG polymer working solution (Beijing Solarbio Science & Technology Co., Ltd.) was added for 10 min incubation at room temperature. Then, the sections were stained with DAB (Jiangsu Kaiji Biology Co., Ltd, China) and hematoxylin and observed under an optical microscope. For immunofluorescence, the sections were deparaffinized to water and fixed in 4% paraformaldehyde for 30 min at room temperature. Then, the sections were sealed with goat serum and incubated with primary antibodies CGRP (14959 S, Cell Signaling Technology, USA, 1:400), SP (S8305, Sigma, USA, 1:5000), and NF-κB P65 (ab16502, AbCam, USA, 1:100), followed by conjugated with anti-rabbit IgG (4412 S, Cell Signaling Technology, USA, 1:500). Images were captured under a laser scanning confocal microscope (Olympus Corporation, Japan).
3
Immunohistochemical Analysis of Neural Markers
4
Evaluation of Chondrocyte Protein Markers
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Total protein was extracted from chondrocytes/cartilage tissue using radio-immunoprecipitation assay lysis buffer containing 1 mM phenylmethanesulfonyl fluoride. Protein concentrations were measured using a bicinchoninic acid assay. Equal amounts of protein (20 μg/lane) were separated by 10% SDS-PAGE and transferred to polyvinylidene difluoride membranes. The membranes were blocked with 5% non-fat milk and incubated with primary antibodies against TLR4, Myd88, IKK-β, β-actin, aggrecan, MMP-3, MMP-9 (ab13556, ab2064, ab124957, ab8227, ab36861, ab52915, ab76003, AbCam, USA, 1:1000, respectively), MMP-13 (ab39012, AbCam, USA, 1:5000), ADAMTS-4 (ab150370, AbCam, USA, 1:1000), ADAMTS-5 (A02802-1, Boster, China, 1:500), CGRP (14959 S, Cell Signaling Technology, USA, 1:1000), SP (S8305, Sigma, USA, 1:2000), NF-κB p65 (ab16502, AbCam, USA, 1:5000), and GAPDH (ab181602, AbCam, USA, 1:10,000) overnight at 4 °C, followed by incubation with horseradish peroxidase-conjugated secondary antibody IgG (ab6721, AbCam, USA) at room temperature. Immune reactivity was visualized using an enhanced chemiluminescent reagent (Pierce; Thermo Fisher Scientific, Inc.). Band intensity was quantitatively analyzed by densitometric analysis on Image J software version 1.37 (National Institutes of Health, Bethesda, MD, USA).
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