Tissue was fixed in 4% paraformaldehyde overnight at 4°C. Tissues were cryoprotected in 30% sucrose/PBS then mounted in optimum cutting temperature medium. Tissue sections of 10 μm were cut on a Cryostat (Leica CM1860), incubated with blocking buffer (Dako, UK) for 1 h, and primary antibodies were applied overnight (4°C): TRPM8 (1:200; Alomone Labs; ACC‐049), CD64 (1:500; Dako; C727801‐2). Tissues were washed (PBS; 3 × 5 min) and species‐specific secondary antibodies conjugated to Alexa Fluor fluorescent dyes (1:400; Thermo Fisher Scientific, UK) applied for 1 h, before washing (PBS; 3 × 5 min), mounting (Vectashield® hard set mounting media; Vector Laboratories, USA) and cover‐slipping. Immunoreactivity of sections was visualized on a Leica DM4000 epi‐fluorescence microscope and images were captured using MetaMorph software (Molecular Devices, UK).
Trpm8
Manufactured by Alomone
Sourced in Israel
TRPM8 is a calcium channel protein that responds to cold temperatures and chemical stimuli. It is involved in the detection and transduction of cold sensations.
Automatically generated - may contain errors
Lab products found in correlation
Trpv1, by Alomone (2 mentions)
Trpa1, by Alomone (2 mentions)
Blocking buffer, by Agilent Technologies (1 mentions)
Vectashield hardset mounting media, by Vector Laboratories (1 mentions)
Alexa fluor fluorescent dyes, by Thermo Fisher Scientific (1 mentions)
Cryostat, by Leica (1 mentions)
Metamorph software, by Molecular Devices (1 mentions)
Fitc goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Lsrii flow cytometer, by BD (1 mentions)
3 3 diaminobenzidine (dab), by Agilent Technologies (1 mentions)
Gapdh, by Beyotime (1 mentions)
4 protocols using trpm8
1
Immunofluorescence Analysis of TRPM8 and CD64
2
Quantifying Platelet Activation Markers
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PRP diluted in PBS was incubated with CD41a (anti-human, APC, Invitrogen) and TRPM8 antibody (1:250 dilution, #ACC-049, Alomone) for 30 minutes at room temperature, and followed by the incubation with the secondary antibody (FITC- goat anti-rabbit IgG, 1:250 dilution, #L43001 Invitrogen) for 30 minutes at room temperature. The samples were fixed with PFA (final concentration 1%).
Samples were run on the LSR II flow cytometer (BD Biosciences). For each sample, a total of 100,000 events were acquired. Data were analyzed by FlowJo V10 software. Platelets were distinguished from other blood cells by using forward (FSC) and side scatter (SSC) and CD61 positive events. The gating strategy for PAC-1 and P-selectin was selected based on the binding of the appropriate isotype antibodies.
Samples were run on the LSR II flow cytometer (BD Biosciences). For each sample, a total of 100,000 events were acquired. Data were analyzed by FlowJo V10 software. Platelets were distinguished from other blood cells by using forward (FSC) and side scatter (SSC) and CD61 positive events. The gating strategy for PAC-1 and P-selectin was selected based on the binding of the appropriate isotype antibodies.
3
Immunohistochemical Staining of Sensory Receptors
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The immunohistochemical staining protocol was carried out following the protocol of our previous study [26 (link)]. Primary antibodies: TRPV1 (rabbit polyclonal antibody, 1 : 100, Alomone Labs, Israel), TRPA1 (rabbit polyclonal antibody, 1 : 100, Alomone Labs, Israel), and TRPM8 (rabbit polyclonal antibody, 1 : 100, Alomone Labs, Israel); and secondary antibody (goat-anti-rabbit 1 : 100, Boster, China) was used and stained with DAB (Dako, Denmark). The percentage of immunopositive cells was analyzed by counting the number of immunopositive neurons and multiplying by 100/number of total number of neurons. A total of 2 DRGs were measured in each animal, and the number of immunoreactive neuronal profiles was counted in a blinded fashion. The percentage of immunopositive cells was used for statistic index.
4
Expression of Thermosensitive TRP Channels
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Mice were sacrificed by cervical dislocation under ether anesthesia 15 days after the WTD and ibuprofen administration. Plantar surfaces of injured hind paw skin were dissected and stored at −80°C for western blot analysis. The expression of TRPV1, TRPA1, and TRPM8 in hind paw skins tissues was determined. The western blot protocol and semiquantitative analysis were carried out following the protocol of our previous study [26 (link)]. The following primary antibodies were used: TRPV1 (rabbit polyclonal antibody, dilution 1 : 100, Alomone Labs, Israel), TRPA1 (rabbit polyclonal antibody, dilution 1 : 100, Alomone Labs, Israel), TRPM8 (rabbit polyclonal antibody, dilution 1 : 100, Alomone Labs, Israel), and GAPDH (internal control, rabbit polyclonal antibody, dilution 1 : 1000, Beyotime, China). A goat-anti-rabbit antibody conjugated to horseradish peroxidase (1 : 5000, Thermo Fisher Scientific, USA) was used as the second antibody. TRPV1, TRPA1, and TRPM8 protein levels were normalized against GAPDH. All experiments were done for four times.
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