Frozen kidney tissues and cells were transferred to 4% paraformaldehyde and fixed at 4 °C overnight. Cells and 4-μm tissue sections were permeabilized with 0.3% Triton X-100 for 10 min and blocked with 5% donkey serum for 1 h. They were then incubated with the following primary antibodies: anti-MAD2B (1:200, ab180579, Abcam, Cambridge, MA, USA), Numb (1:50, sc-136554, Santa Cruz, CA, USA), anti-Synaptopodin (1:200, #163004, Synaptic Systems, Gottingen, Germany), and anti-Nephrin (1:100, AF3159, R&D, Minneapolis, MN, USA) overnight at 4 °C. Alexa Fluor 488 IgG and Alexa Fluor 594 IgG (1:200, Invitrogen, Carlsbad, CA, USA) were used as secondary antibodies. Nucleus was counterstained with Hoechst (Beyotime Biotechnology, Shanghai, China). Sections were observed under fluorescence microscope and images were captured at identical microscopic settings.
Anti synaptopodin
Manufactured by Synaptic Systems
Sourced in United States, Germany
Anti-Synaptopodin is a research-use only antibody product. It is designed to detect the synaptopodin protein, which is a structural protein found in certain types of cells. The antibody can be used in various cell biology research applications, such as immunofluorescence or western blotting, to study the expression and localization of synaptopodin.
Automatically generated - may contain errors
Lab products found in correlation
Anti nephrin, by R&D Systems (2 mentions)
Alexa fluor 488 igg, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 594 igg, by Thermo Fisher Scientific (2 mentions)
Hoechst, by Beyotime (2 mentions)
Af3159, by R&D Systems (1 mentions)
Ab180579, by Abcam (1 mentions)
Alexa 488, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by New England Biolabs (1 mentions)
Ds ri1 camera, by Nikon (1 mentions)
Alexa 568 conjugated goat anti rabbit, by Thermo Fisher Scientific (1 mentions)
Axioplan 2, by Zeiss (1 mentions)
4 protocols using anti synaptopodin
1
Immunostaining of Kidney Tissues and Cells
2
Immunofluorescence Labeling of Brain Sections
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Free-floating sections were washed in TBS, blocked with 5% bovine serum albumin (BSA; New England BioLabs, Ipswich, MA, USA) containing 0.1% Triton X-100 for 1 h at room temperature to reduce non-specific staining and incubated in primary antibody solution containing 2% BSA, 0.25% Triton X-100, 0.1% NaN3 in 0.1 M TBS for 48 h at room temperature. The following primary antibodies were used: anti-βIV-spectrin (rabbit, 1:500; selfmade, see Schlüter et al., 2017 (link)), anti-synaptopodin (rabbit, polyclonal, 1:1,000; Synaptic Systems, Göttingen, Germany) and anti-GFP488 (mouse, 1:500, fluorescence-labeled Alexa 488; Sigma-Aldrich). For immunofluorescence detection, sections were incubated with secondary fluorescence-labeled antibodies (1:1,000; Alexa 488, 568, 633; Vector Labs., Burlingame, CA, USA) for 24 h at room temperature.
3
Kidney Immunofluorescence Staining in Mice
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Kidney sections of experimental mice were prepared and indirect immunofluorescence staining was performed as described previously [38 (link), 39 (link)]. The following primary antibodies were used: anti-AEP (Santa Cruz, Cat No. sc-133234, 1:50), anti-Synaptopodin (Synaptic Systems, Cat No. 163004, 1:200), anti-Nephrin (R&D, Cat No. AF3159, 1:100), and anti-cofilin-1 N138 (1:50). The secondary antibodies: Alexa Fluor 488 IgG (Invitrogen, Cat No. S11223, 1:200) and Alexa Fluor 594 IgG (Invitrogen, Cat No. A10438, 1:200). Nuclear was counterstained with Hoechst (Beyotime Biotechnology, Cat No. C1011).
4
Quantifying Synaptopodin in Spinal Cord
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immunodetection of synaptopodin was performed on spinal cord cryostat sections (30 µm thickness). Sections were incubated for 72 hours at 4 °C with anti-synaptopodin (1/250; Synaptic Systems) and anti-GFP (1/500; AveLabs). After rising, sections were incubated with Alexa 568-conjugated goat anti-rabbit (1/500; Molecular probes) and Alexa 488-conjugated goat anti-chicken (1/200; ThermoFisher) for 2 hours at room temperature.
Image acquisition was performed with an epifluorescent microscope AxioPlan 2 (Zeiss) and a DsRi1 camera (Nikon) using the 10x objective, and the rhodamine and FITC filters.
To quantify the percentage of the area labeled for synaptopodin, we developed a macro on image j software. On the other hand, to quantify synaptopodin fluorescence intensity, we focused only on the cells expressing the ShRNA construct as determine by the expression of GFP. Then we applied the corrected total cell fluorescent method11 (link). Briefly, this method quantifies the intensity of fluorescence of each cell after subtraction of the fluorescence background and correction by the area of the ROI.
Image acquisition was performed with an epifluorescent microscope AxioPlan 2 (Zeiss) and a DsRi1 camera (Nikon) using the 10x objective, and the rhodamine and FITC filters.
To quantify the percentage of the area labeled for synaptopodin, we developed a macro on image j software. On the other hand, to quantify synaptopodin fluorescence intensity, we focused only on the cells expressing the ShRNA construct as determine by the expression of GFP. Then we applied the corrected total cell fluorescent method11 (link). Briefly, this method quantifies the intensity of fluorescence of each cell after subtraction of the fluorescence background and correction by the area of the ROI.
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!