We have previously demonstrated that in PD, nigral neurons with α-syn inclusions lose tyrosine hydroxylase expression relative to neighbors without inclusions or age-matched controls.18 (link) Thus, to see whether subjects with MMF display a similar phenomenon, a double-label immunofluorescence procedure was employed to determine whether α-syn inclusions affected dopaminergic neuronal expression in these cases. Midbrain sections from all three groups were incubated in the first primary antibody p-S129-α-syn (1:1000) overnight and the goat anti-rabbit antibody coupled to DyLight 649 (1:200, Jackson ImmunoResearch) for 1 h. After blockade for 1 h, the sections were then incubated in the second primary antibody (TH, 1:5000) overnight, and the goat anti-mouse antibody coupled to DyLight 488 (1:200) for 1 h. The sections were mounted on gelatin-coated slides, dehydrated through graded alcohol, cleared in xylene, and covered using DPX (Sigma-Aldrich).
Dylight 488
Manufactured by Merck Group
Sourced in Australia
DyLight 488 is a fluorescent dye produced by Merck Group. It is designed for use in various laboratory applications that require a specific excitation and emission wavelength profile.
Automatically generated - may contain errors
Lab products found in correlation
Dibutylphthalate polystyrene xylene (dpx), by Merck Group (1 mentions)
Dylight 649, by Jackson ImmunoResearch (1 mentions)
Fluorescence mounting medium, by Agilent Technologies (1 mentions)
Igg free bovine serum albumin, by Merck Group (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Goat serum, by Merck Group (1 mentions)
Antigen unmasking solution, by Vector Laboratories (1 mentions)
Vectastain abc kit, by Vector Laboratories (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Ab955, by Abcam (1 mentions)
3 protocols using dylight 488
1
Evaluating Dopaminergic Neuron Changes in Multisystem Mitochondrial Failure
2
Sequential Immunostaining of GFAP and p75NTR
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Sections were fixed in methanol at 4°C for 3 min, and then in acetone at 4°C for 3 min. All the following steps were performed at room temperature. The sections were airdried for 1 h and then rehydrated for 10 min in phosphate-buffered saline (PBS). All washes (3x10 min) between stages were performed in PBS. After the sections had been permeabilized with 0.2% Triton X-100 (Sigma-Aldrich) in PBS for 5 min, potential non-specific binding sites were blocked with antibody dilution buffer [2% goat serum (Sigma-Aldrich) and 1% IgG-free bovine serum albumin (Sigma-Aldrich) in PBS] for 20 min at room temperature. Sections were then incubated with primary antibodies overnight at 4°C. After being washed, the sections were then incubated with secondary antibodies for 1 hour at room temperature. Following the final washing step, sections were mounted with Fluorescence Mounting Medium (Dako, North Sydney, Australia).
For immunostaining with two rabbit antibodies [anti-glial fibrillary acidic protein (GFAP) and anti-p75 neurotrophin receptor (p75NTR)] on the same section, a sequential immunostaining approach30 (link) was used [anti-GFAP+goat anti-rabbit IgG DyLight 488+ rabbit IgG (Sigma) + (mixture of anti-p75NTR +goat anti-rabbit IgG Alexa 555; incubated half hour before application)].
For immunostaining with two rabbit antibodies [anti-glial fibrillary acidic protein (GFAP) and anti-p75 neurotrophin receptor (p75NTR)] on the same section, a sequential immunostaining approach30 (link) was used [anti-GFAP+goat anti-rabbit IgG DyLight 488+ rabbit IgG (Sigma) + (mixture of anti-p75NTR +goat anti-rabbit IgG Alexa 555; incubated half hour before application)].
3
Kidney Injury Molecule-1 Immunohistochemistry
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Renal biopsy tissue was fixed in 4% paraformaldehyde for paraffin-embedded kidney sections (4 µm), which were then deparaffinized and rehydrated for the following staining techniques. For histologic examination, H&E staining was performed using the standard methods. Briefly, rehydrated kidney sections were subjected to antigen retrieval using the Antigen Unmasking Solution purchased from Vector, followed by blocking with 2% normal goat serum. The sections were then incubated with primary antibodies at the following dilutions: KIM-1(1:200) (MA5-43897, Thermo Fisher Scientific, US), CD4 (1:100) (ab231460, Abcam, US), CD68 (1:100) (ab955, Abcam, US) overnight at 4 °C, washed three times in PBS, incubated with appropriate secondary antibodies, and washed with PBS again. For immunohistochemistry, the signals were visualized using VECTASTAIN ABC kits (Vector), followed by counterstaining with hematoxylin and capturing images using a CX31 microscope with a DP73–1-51 digital camera (Olympus). For immunofluorescence staining, after incubation with the primary antibodies indicated and washing with PBS, the sections were incubated with DyLight 488-conjugated secondary antibodies at 37 °C for 1 h. Nuclei were counterstained with DAPI (1:1000; Sigma-Aldrich). Images were captured by the Vectra Imaging System. KIM-1 positive area in glomeruli was measured by the Image J software.
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