Streptavidin alexa fluor 633 conjugate

Mice were perfused 21 days’ post-induction (dpi) with 4% paraformaldehyde (PF) in a phosphate buffer (PB). They were then post-fixed for over 2 h in the same solution and stored at 4 °C in PBS. Coronal vibratome sections (50 µm) were washed and permeabilized three times with 0.5% Triton X-100 (PBS-T), washed three times in 0.1% PBS-T, and blocked for 30 min at room temperature (RT) with 5% normal goat serum (NGS, S26-100ML: Merck-Millipore). Brain sections were incubated overnight at 4 °C with the following antibodies in 5% NGS and 0.1% PBS-T: rabbit anti-PDGFRα (1:300, 3174S: Cell Signaling) and biotinylated tomato lectin (TL, 1:50, L0651: Sigma-Aldrich). After washing the brain slices three times with 0.1% PBS-T, they were incubated for 2 h at RT with a secondary antibody coupled to Alexa 633 (1:1000, Invitrogen) or a Streptavidin–Alexa Fluor 633 conjugate (1:1000, S21375: Invitrogen Life Technologies (Carlsbad,. CA, USA ). Prior to visualization, they were washed 6 times in 0.1% PBS-T and then 1× PBS.

Pharmacological inhibitors were prepared in either DMSO or 20 % acetonitrile/water according to the manufacturer’s recommendations and used at the indicated concentrations. EIPA, CPZ, Gen, and Rot were purchased from Sigma Aldrich. The Rac1 inhibitor W56 was purchased from Tocris Bioscience.
Specific antibodies including mouse anti-beta actin (ab8226), rabbit anti-EEA1 antibody (ab2900), Anti-LAMP1 [H4A3], anti-beta actin antibody [AC-15], anti-beta tubulin antibody, anti-neuron specific beta III tubulin were purchased from Abcam. Alexa Fluor 488 goat anti-mouse, Alexa Fluor 488 goat anti-rabbit, streptavidin Alexa Fluor 633 conjugate, streptavidin Alexa Fluor 488 conjugate, Alexa Fluor 488 donkey anti-sheep, Alexa Fluor 488 donkey anti-rabbit, SYTOX Red dead cell stain, FM® 1-43FX fixable analogue of FM® 1–43 membrane stain were purchased from Invitrogen Life Technologies. Donkey anti-sheep/goat IgG HRP conjugate and goat anti-mouse IgM + IgG + IgA (H + L) HRP conjugates were purchased from Millipore.
Sheep anti-SOD1 was purchased from Thermo Fisher Scientific. Mouse monoclonal anti-human TARDBP antibody (clone k1B8) was purchased from Abnova. Anti-BiP/GRP78 was purchased from BD Transduction Laboratories. FITC-conjugated sheep anti-mouse was purchased from Silenus. RedDot 2 was obtained from Biotium. Goat Anti-Rabbit IgG (H + L)-HRP Conjugate was obtained from Bio-Rad.