Mice were perfused transcardially with 4% paraformaldehyde under isoflurane anesthesia, and L3-L5 DRG tissues were collected. After being cryprotected with 30% sucrose, the tissues were then sectioned to a thickness of 15μm and were post-fixed with 4% paraformaldehyde. DRG sections were then incubated with blocking solution containing 0.1% Triton X-100, 3% BSA, and 0.02% sodium azide in phosphate-buffered saline for 2 h at room temperature, followed by incubation at with the anti-TNF-α antibody (1:300; ab1793, Abcam, Shanghai, China) prepared in blocking solution at 4°C overnight. The fluorescent-labeled secondary antibodies (Abcam, 1:200) specific to the IgG species were then used. All the fluorescent imaging was performed with the same laser power and exposure time by a CKX41 microscope with an Olympus U-RFLT50 Power Supply Unit (Olympus, Tokyo, Japan). The images were quantitatively analyzed by NIH ImageJ.
U rflt50 power supply unit
Manufactured by Olympus
Sourced in Japan
The U-RFLT50 Power Supply Unit is a compact and reliable power supply designed for laboratory equipment. It provides a stable and adjustable output voltage to power various devices used in research and analysis. The unit features overload protection and is compatible with a range of Olympus laboratory instruments.
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5 protocols using u rflt50 power supply unit
1
Immunohistochemical Analysis of TNF-α in DRG
2
Immunohistochemical Analysis of Tissue Samples
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Mice were anesthetized with isoflurane and then perfused transcardially with 4% paraformaldehyde. The tissue samples were cryprotected with 30% sucrose. The tissues were cut to a thickness of 15 μm and were post-fixed briefly with 4% paraformaldehyde and then incubated with blocking solution containing 3% BSA, 0.1% Triton X-100, and 0.02% sodium azide in PBS for 2 h at room temperature. After blocking, the sections were incubated at 4 °C overnight with the primary antibodies prepared in blocking solution. The secondary antibody was goat anti-rabbit (1:500) antibody (Molecular Probes, Carlsbad, CA, USA). We incubated the slices with fluorescence-conjugated secondary antibodies or avidin-biotin horseradish peroxidase complex (1 h), washed them three times with 0.1 M Tris buffer (5 min each), and then developed them in diaminobenzidine tetrahydrochloride (1–2 min), before washing three times with 0.1 M Tris buffer (5 min each). Finally, the sections were incubated with 0.1 M Tris buffer to stop the reaction. The slides were mounted with cover slips and visualized by using a CKX41 microscope with an Olympus U-RFLT50 Power Supply Unit (Olympus, Tokyo, Japan).
3
Immunohistochemical Analysis of SIRT5 Expression
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Mice were anesthetized with an overdose of choral hydrate and with the cervical dislocation execution. The right ankle joint of mice was taken and samples collected were decalcified in 13% EDTA (pH 7.3), then placed in 30% sucrose overnight and embedded in OCT at −20 °C the next day. Frozen sections were cut (30 μm) and placed on glass micro slides coated with APS. Sections were fixed in 4% paraformaldehyde and incubated in a blocking solution containing 3% BSA, 0.02% Na N30.1%, and Triton X-100 in PBS for 2h at room temperature. After blocking, sections were incubated with the appropriate primary antibodies in a blocking solution at 4 °C overnight. The primary antibodies used were: anti-SIRT5 (1:400) from ProteinTech. The secondary antibody was a goat anti-rabbit (1:500) antibody (ProteinTech Group, Chicago, IL, USA). Slides were mounted with coverslips and visualized using a fluorescence microscope (CKX41 with an Olympus U-RFLT50 Power Supply Unit; Olympus, Tokyo, Japan). Image-pro plus 6.0 (Media Cybernetics, Inc., Rockville, MD, USA) was applied to analyze the integrated optic density (IOD) and pixel area (AREA), and calculated the average optical (AO), AO = IOD/AREA.
4
Immunofluorescence Analysis of TRPV1 and pERK
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Mice were anesthetized with 1% isoflurane and intracardially perfused, first with normal saline followed by 4% paraformaldehyde. Mice brains were then placed in 30% sucrose and embedded in tissue optimum cutting temperature (OCT)-freeze medium at –20°C on the following day. Frozen sections were cut (20 μm) and placed on amino propyltriethoxy silane (APS)-coated glass microslides. Subsequently, the sections were post-fixed in 4% paraformaldehyde for 3 min and incubated in blocking solution containing 3% bovine serum albumin (BSA, Merck, USA), 0.1% Triton X-100, and 0.02% NaN3 in phosphate buffered saline (PBS) for 2 h at room temperature. After blocking, brain sections were incubated with primary antibodies in a blocking solution at 4 °C overnight. The following primary antibodies were used: anti-TRPV1 (1:500, Alomone, Israel) and anti-pERK (1:500, Alomone, Israel) from Alomone. The secondary antibody was a goat anti-rabbit (1:500) antibody (MolecularProbes, Carlsbad, CA, USA). Slides were mounted with cover slips and visualized using a fluorescence microscope (CKX41 with an Olympus U-RFLT50 Power Supply Unit, Olympus, Tokyo, Japan).
5
Immunohistochemical analysis of pNR1 in the mouse spinal cord
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Mice were anesthetized with isoflurane and then perfused transcardially with 4% paraformaldehyde. Next, we dissected the SC from the lumbar section. The tissue samples were embedded in paraffin. The paraffin-embedded sections were cut to a thickness of 5 μm and pasted onto micro-slide glasses coated with APS. The sections were post-fixed briefly with 4% paraformaldehyde for 3 min and then incubated with blocking solution containing 3% BSA, 0.1% Triton X-100, and 0.02% sodium azide in PBS for 2 h at room temperature. After blocking, the sections were incubated at 4°C overnight with the primary antibodies against anti-pNR1 ( 1 ). The secondary antibody was goat anti-rabbit (1:500) antibody (Molecular Probes, Carlsbad, CA, USA). We incubated the slices with avidin-biotin horseradish peroxidase complex (1 h), washed them three times with 0.1M Tris buffer (5 min each), and then developed them in diaminobenzidine tetrahydrochloride (1-2 min), before washing three times with 0.1 M Tris buffer (5 min each). Finally, the sections were incubated with 0.1 M Tris buffer to stop the reaction. The slides were mounted with cover slips and visualized by using a CKX41 microscope with an Olympus U-RFLT50 Power Supply Unit (Olympus, Tokyo, Japan).
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