Sagittal kidney tissue sections (4 μm thick) and HK-2 cells seeded on chamber slides (Thermo Scientific) and incubated with treatments as described previously were prepared for immunofluorescence staining and in situ fluorescent TUNEL staining. First, the sections and cells were fixed with 4% paraformaldehyde (Sigma-Aldrich), followed by permeabilization in 0.1% Triton X-100 and incubated with 5% BSA (Sigma-Aldrich). The slides and cells were then incubated with anti-RIP3 monoclonal antibody (#95702, Cell Signaling Technologies, Danvers, MA, USA; 1:100 dilution) or anti-RIP3 polyclonal antibody (ab152130, Abcam, Cambridge, MA, USA; 1:100 dilution) overnight at 4°C and then incubated with Alexa Fluor 555-conjugated goat anti-rabbit IgG (H+L) (#4413, Cell Signaling Technologies, Danvers, MA, USA; 1:200 dilution). After rinsing 3 times in 0.1 M PBS (pH 7.4), the samples were incubated with in situ cell death detection kit reagents (Fluorescein, Roche, Basel, Switzerland) according to the manufacturer’s instructions and counterstained with 4',6-diamidino-2-phenylindole (DAPI). Finally, the images were captured by confocal microscopy (LEICA TCS SP2, Wetzlar, Germany), and the cell counting was performed by a pathologist blinded to the experimental conditions.
Alexa fluor 555 conjugated goat anti rabbit igg h l
Manufactured by Cell Signaling Technology
Sourced in United States
Alexa Fluor 555-conjugated goat anti-rabbit IgG (H+L) is a secondary antibody that binds to the heavy and light chains of rabbit immunoglobulin G (IgG). The antibody is conjugated with Alexa Fluor 555, a fluorescent dye that can be detected using appropriate instrumentation.
Automatically generated - may contain errors
Lab products found in correlation
In situ cell death detection kit reagents, by Roche (1 mentions)
Chamber slide, by Thermo Fisher Scientific (1 mentions)
Anti rip3 monoclonal antibody, by Cell Signaling Technology (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
F ab 2 fragment, by Cell Signaling Technology (1 mentions)
Bz x710 fluorescence microscope, by Keyence (1 mentions)
Cryostat, by Leica (1 mentions)
3 protocols using alexa fluor 555 conjugated goat anti rabbit igg h l
1
Immunofluorescence and TUNEL Staining of Kidney Tissue and Cells
2
Dual Immunofluorescence Staining for Colitis
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Double immunofluorescence staining for human colitis tissue of UC patients, normal colon tissue of healthy volunteers, and murine colitis tissue was performed. Frozen sections were treated with citrate buffer (pH 6.0) for 10 min at 100 °C for antigen retrieval and then blocked with blocking solution for 30 min at room temperature. Sections were incubated with a mixture of 2 primary antibodies overnight at 4 °C. The sections were then incubated with a mixture of 2 secondary antibodies for 1 h at room temperature. The following secondary antibodies were applied: Alexa Fluor 488-conjugated goat anti-mouse IgG (H + L), F(ab’)2 Fragment (#4408, Cell Signaling Technology, Danvers, MA), Alexa Fluor 555-conjugated goat anti-rabbit IgG (H + L), F(ab’)2 Fragment (#4413, Cell Signaling Technology), and Alexa Fluor 488-confugated goat anti-rat IgG (H + L) (#4416, Cell Signaling Technology). Nuclei were counterstained with 4’,6-diamidino-2-phenylindole (DAPI). The stained sections were observed using a BZ-X710 fluorescence microscope (Keyence, Osaka, JPN).
3
Immunofluorescence Analysis of Regenerated Nerves
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The regenerated nerves were fixed with 4% paraformaldehyde for 24 hours and cryoprotected with increasing concentrations of sucrose (10%, 20%, and 30%). The samples were embedded in optimal cutting temperature compound and cut into 12 μm-thick sections using a cryostat (Leica). The sections were permeabilized with 0.2% Triton X-100, blocked with 10% goat serum, and incubated at 4°C overnight with rabbit anti-S100 antibody (1:400; Sigma-Aldrich, Cat# S2644, RRID: AB_477501) and mouse anti-neurofilament (NF200; 1:400; Sigma-Aldrich, Cat# N0142, RRID: AB_477257) antibody or rabbit anti-CD31 (1:400; Sigma-Aldrich, Cat# SAB4503968, RRID: AB_2895674) antibody. After rinsing with PBS, the sections were incubated with Alexa Fluor 555-conjugated goat anti-rabbit IgG H+L (1:2000; Cell Signaling Technology, Cat# 4413, RRID: AB_10694110; Beverly, MA, USA,) or Alex Fluor 488-conjugated goat anti-mouse IgG H+L (1:2000; Cell Signaling Technology Cat# 4408, RRID: AB_10694704) in the dark for 1 hour at room temperature. Finally, the sections were stained with DAPI, rinsed with PBS, and observed using a fluorescence microscope. The percentages of NF200- and S100-positive cells were analyzed with Image-Pro Plus 6.0 software. The microvessel density was determined by counting the number of microvessels in each microscopic field.
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