Double-label immunofluorescence was performed to visualize colocalization of p-α-syn with subtypes of ENS neurons including catecholaminergic (tyrosine hydroxylase; TH) and vasoactive intestinal peptide (VIP)-ergic and to visualize colocalization of α-syn and PGP9.5 (see Table 2 for full list of primary antibody information). Slides were deparaffinized and treated for heat antigen retrieval in a microwave for 6 mins at 100% power followed by 3 mins at 60% power and left to cool for 30 mins at room temperature. Tissue was blocked with 5% donkey serum and 2% BSA solution and incubated in primary antibodies overnight at 4°C. The sections were then incubated with Alexa Fluor-conjugated secondary antibody (1:1000) against the appropriate species and cover slipped with mounting medium with DAPI (Vector Laboratories, Burlingame, CA). Negative controls were performed in parallel by omitting the primary antibodies.
Alexa fluor conjugated secondary antibody
Manufactured by Vector Laboratories
Sourced in Canada, United States
Alexa Fluor-conjugated secondary antibodies are fluorescent-labeled antibodies used for detection in immunoassays. They are designed to bind and detect primary antibodies, amplifying the signal for improved sensitivity. These antibodies are available with a variety of Alexa Fluor dyes, each with distinct fluorescent properties.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (4 mentions)
Ultraview vox spinning disk confocal microscope, by PerkinElmer (1 mentions)
Te2000 microscope, by Nikon (1 mentions)
Gap43, by Merck Group (1 mentions)
A1 confocal microscope, by Nikon (1 mentions)
Tcs sp5 microscope, by Leica (1 mentions)
Vectashield, by Vector Laboratories (1 mentions)
6 protocols using alexa fluor conjugated secondary antibody
1
Double-Label Immunofluorescence for Alpha-Synuclein
2
Immunocytochemistry and Alkaline Phosphatase Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were fixed with 4% formaldehyde in PBS for 5 min, permeabilized with 1% Triton X-100 in PBS for 10 min, and blocked with 5% BSA in PBS for 1 h. Cells were then stained with appropriate primary antibody and AlexaFluor-conjugated secondary antibody (Vector Laboratories, USA). Pictures were taken with the UltraVIEW VoX Spinning Disk Confocal Microscope (PerkinElmer, USA).
Alkaline phosphatase staining was performed using the Alkaline Phosphatase Staining Kit (Vector Laboratories, USA) following the manufacturer's instructions. Pictures were taken using a TE2000 microscope (Nikon, Japan) and the quantification of alkaline phosphatase-positive colonies was performed as described [39] (link).
Alkaline phosphatase staining was performed using the Alkaline Phosphatase Staining Kit (Vector Laboratories, USA) following the manufacturer's instructions. Pictures were taken using a TE2000 microscope (Nikon, Japan) and the quantification of alkaline phosphatase-positive colonies was performed as described [39] (link).
3
Cardiac Nerve Regeneration Imaging
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immunofluorescence stainings were performed in cardiac tissue to identify catecholaminergic phenotype (by colocalization of TH-immunoreactivity (ir) and PGP9.5-ir) and sympathetic re-innervation (by TH-ir and growth associated protein 43 (GAP43)-ir) in cardiac nerve bundles. Slides were deparaffinized and antigen retrieval performed as described above. Tissue was blocked with 5% donkey serum and 2% BSA solution and incubated in primary antibodies GAP43 (1∶100; Millipore; Billerica, MA), PGP9.5 (1∶100), and TH (1∶200) for 2 days in 4°C. The sections were then incubated with alexafluor-conjugated secondary antibody (1∶1000) against the appropriate species and coverslipped with mounting media with DAPI (Vector Laboratories, Burlingame, CA). Brain tissue from a rhesus fetus at embryonic day 38 was used as a positive control for GAP43.
4
Immunofluorescence Staining of Neuronal Markers
5
Triple-Label Immunofluorescence of Neurodegeneration
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Triple-label immunofluorescence was performed to assess distribution of the proteins α-syn, p-α-syn, tau and p-tau in the ENS ganglia and nerve fibers identified by the panneuronal marker PGP9.5. Slides were deparaffinized and treated for heat antigen retrieval in a microwave for 6 mins at 100% power followed by 3 mins at 60% power and left to cool for 30 mins at room temperature. Tissue was incubated with 5% donkey serum and 2% BSA solution, followed by primary antibodies (Table 2 ) overnight at 4°C. The sections were then incubated with Alexa Fluor-conjugated secondary antibody (1:1000) against the appropriate species and coverslipped with mounting medium with 4′,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, Burlingame, CA). Negative controls were performed in parallel by omitting the primary antibodies. Confocal images were obtained using a Nikon A1 confocal microscope (Tokyo, Japan).
6
Analyzing Lysosomal Trafficking in MEFs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Around 1.5 • 10 5 MEFs were seeded in 12-well plates on Alcian blue coverslips, transfected as previously described, and incubated with/without 50 nM BafA1. After 12 h, cells were washed twice with PBS and fixed in 3.7% formaldehyde for 20 min at room temperature (RT). Then, cells were washed three times with PBS and permeabilized using a solution composed of 10% goat serum, 15 mM glycine, 0.05% saponin, and 10 mM HEPES in PBS (PS) for 15 min at RT. After permeabilization, cells were incubated with the anti-IDS diluted 1:100 and anti-LAMP1 diluted 1:50 in PS for 90 min at RT. Subsequently, cells were washed three times (5 min each) in PS, supplemented with Alexa Fluor-conjugated secondary antibody diluted 1:300 in PS for 40 min, washed with PS and water, and mounted with Vectashield (Vector Laboratories, Burlingame) containing 4¢,6-diamidino-2phenylindole (DAPI). Pictures were collected by Leica TCS SP5 microscope with a 63 • /1.4 N.A. objective (Leica HCX PL APO lambda blue 63.0 • 1.40 OIL UV, Wetzlar, Germany).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!