Alexa fluor 568 dye

Mouse retinas were dissected, fixed, and cryosectioned at 10 μm thickness by standard protocols as described earlier for immunohistochemistry.²¹ (link) The following antibodies were used: rabbit polyclonal antibody against the N-terminus of RS1 (amino acid residues 24–37 [1:1000; Custom made, ThermoFisher Scientific, Waltham, MA, USA]³³ (link); monoclonal antibody against the protein kinase C-alpha [1:500, Santa Cruz Biotechnology, Dallas, TX, USA]; mouse monoclonal antibody against glutamine synthetase [1:1000; Sigma-Aldrich, St. Louis, MO]; calretinin [1:2000; BD Biosciences, San Jose, CA, USA]; goat anti-mCherry [1:2000; Biorbyt, Cambridge, UK], and 1:1000 mouse monoclonal Na+/K+ ATPase [alpha-3 subunit] antibody [Na/K-ATPase alpha 3; ThermoFisher Scientific], secondary antibody conjugated to Alexa Fluor 568 dye or Alexa Fluor 488 dye [ThermoFisher Scientific]). Retinal images were obtained and processed with a Nikon C2 confocal microscope with Advanced Element software (Nikon, Tokyo, Japan). Image analysis was performed using image-editing software (Photoshop CS6; Adobe Systems, Inc., San Jose, CA, USA).

For immunofluorescence analysis, cells were fixed with 4% paraformaldehyde for 30 min. Subsequently, they were permeabilized and blocked for 30 min with 0.1% Triton X‐100 (#T8787, Sigma‐Aldrich), 10% goat serum (#16210072, Gibco^®, Life Technologies), and 1% BSA (#A9647, Sigma‐Aldrich) in PBS (#20012‐019, Gibco^®, Life Technologies). When AGO1 antibodies were used for staining, the blocking buffer was modified to use donkey serum (D9663‐10ML) instead of goat serum. Thereafter, the cells were incubated with primary antibodies (1:100 dilution) in the same buffer at desired dilution overnight at 4°C. Secondary anti‐rabbit, anti‐goat, or anti‐mouse antibodies labeled either with Alexa Fluor^® 488 dye (green), Alexa Fluor^® 568 dye (orange), Alexa Fluor^® 594 dye (red), or Alexa Fluor^® 647 dye (far red) fluorochromes (Molecular Probes) were used at 1:500 dilutions. The cells were mounted on a glass slide with VECTASHIELD DAPI (Vector Laboratories), and cells were mostly observed and documented with a ZEISS point scanning confocal LSM 700/LSM 800 inverted microscopes with a PLAN APO 40× (NA = 1.3) and 63× (NA = 1.4) oil‐immersion objectives. Laser settings and digital gain settings were set so as to avoid any saturated pixel in the resulting capture. Z‐stack images were captured wherever mentioned in text.

The cortactin (ab33333), PTP1B EP1837Y (ab52650) and PTP1B EP1841Y (ab75856) antibodies were obtained from Abcam (Cambridge, MA). The Tks5 (sc30122) antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA) and labeled with Alexa Fluor 568 dye (Molecular Probes, Eugene, OR) for pY421-cortactin staining experiments as per the manufacturer’s instructions. The phospho-Y421 cortactin (C0739) antibody was from Sigma-Aldrich (St. Louis, MO). The PTP1B inhibitor (539741) was from Calbiochem (San Diego, CA). All secondary antibodies were Alexa Fluor conjugated and obtained from Molecular Probes (Life Technologies, Carlsbad, CA). The Pan-Mena antibody was purchased from Novus Biologicals (Littleton, CO; Cat# NBP1–87914). The antibody specific for Mena^INV used in this study has been described elsewhere59 . pEGFP-PTP1B C3 was a gift from Anna Huttenlocher (University of Wisconsin, Madison, WI). pCAX-eGFP and pCAX-eGFP-Mena^INV vectors were produced by the Gertler lab (MIT, Cambridge, MA)45 (link).

HeLa cells were grown on Labtek chamber slides and transiently transfected with ELOVL4 constructs. After 48 h, slides were rinsed and fixed as per Logan et al. (9 (link)). The slides were blocked with 5% nonfat dry milk and incubated with primary rabbit anti-HA antibody (Clonetech) and mouse anti-calnexin antibody (Abcam) overnight at 4°C. The following day, cells were washed and incubated with secondary anti-rabbit antibody conjugated with Alexa Fluor® 488 dye (Invitrogen) and anti-mouse antibody conjugated with Alexa Fluor® 568 dye (Invitrogen). The slides were then washed and coverslipped with Vectashield with DAPI mounting medium (Vector Labs) and imaged by confocal microscopy (Olympus FluoView 500, Olympus, Melville, NY).

Podocytes grown on Collagen-I coated coverslips were incubated with 0.001–1 ng/mL IL-6 for 1 h. Cells were washed with PBS (× 2), fixed in 4% paraformaldehyde for 10 min and then incubated with phalloidin conjugated Alexa Fluor 568 dye (1:200 dilution, Invitrogen, Carlsbad, CA) for 30 min in the dark. Cover slips were mounted in 5% n-propyl gallate in buffered glycerol (glycerol:PBS, 9:1). Cells were viewed using a Leica confocal microscope (Leica DMI 4000 B) at 561 nm excitation. Laser intensity, gain settings, scaling, individual section depth, and pinhole settings were constant throughout.