Alexa fluor 546 conjugated donkey anti mouse igg

The primary antibodies used for immunohistochemistry in the current study included: mouse anti-neurophysin II (AVP-NP) [1:100; PS41; kindly provided by Dr. Harold Gainer, National Institutes of Health (NIH), Bethesda, MD, USA]^{55 (link),56} , guinea pig anti-AVP (1:2000; T-5048; Peninsula, San Diego, CA, USA), mouse anti-neurophysin I (OT-NP; 1:100; PS38; a gift from Dr. Harold Gainer)^{55 (link),56} , chicken anti-GFP (1:10,000; ab13970; Abcam, San Diego, CA, USA), rabbit anti-IBA1 (1:200; ab178846; Abcam) and rabbit anti-GFAP (1:200; ab7260; Abcam). The following secondary antibodies were used in the present study: for immunofluorescence staining, Alexa Fluor 488-conjugated goat anti-chicken IgY (H + L) (1:1000; A11039; Invitrogen, San Diego, CA, USA), Alexa Fluor 546-conjugated donkey anti-mouse IgG (H + L) highly cross-adsorbed (1:1000; A10036; Invitrogen), Cy3-conjugated affinipure donkey anti-guinea pig IgG (H + L) (1:500; 706-165-148; Jackson ImmunoResearch, West Grove, PA, USA), Alexa Fluor 647-conjugated donkey anti-mouse IgG (H + L) highly cross-adsorbed (1:1000; A31571; Invitrogen) and Alexa Fluor 647-conjugated donkey anti-rabbit IgG (H + L) highly cross-adsorbed (1:1000; A31573; Invitrogen); for immunoelectron microscopy, biotinylated horse anti-mouse IgG (H + L) (1:200; BA-2000; Vector Laboratories, Burlingame, CA, USA).

Mouse monoclonal antibodies (mAbs) against RABV N or P were prepared in our laboratory46 (link). The rabbit polyclonal antibody (pAb) against GAPDH was purchased from Hangzhou Good Here Biotechnology Co. Ltd (Hangzhou, China). Rabbit anti-ß-actin and anti-Myc pAbs were obtained from Hangzhou Huaan Biotechnology Co. Ltd (Hangzhou, China). Rabbit anti-Hsp90AA1, anti-Cdc37 (phospho S13) and anti-Cdc2 mAbs were purchased from Abcam (Cambridge, MA, USA). Rabbit anti-LC3B, anti-Cdc37 and anti-IKKa mAbs were purchased from Cell Signaling Technology (Beverly, MA, USA). Mouse anti-Flag (clone M2) mAb was purchased from Sigma-Aldrich (St. Louis, MO, USA). Alexa Fluor 546-conjugated donkey anti-mouse IgG and Alexa Fluor 647-conjugated goat anti-rabbit IgG were purchased from Invitrogen Life Technologies (Carlsbad, CA, USA). The proteasome inhibitor MG-132 and the de novo protein synthesis inhibitor cycloheximide (CHX) were purchased from Beyotime Biotechnology (Shanghai, China). The Hsp90 inhibitor 17-(Allylamino)-17-demethoxygeldanamycin (17-AAG), the Hsp90 allosteric inhibitor celastrol and the autophagy inhibitor wortmannin were purchased from Sigma.

To visualize MTOC of activated/expanded Vβ2⁺ CD8 T_EM and Treg transmigrating on TNF-activated CIITA HDMEC plus TSST-1, samples were stained with anti-Vβ2TCR mAb (Beckman Coulter), Alexa Fluor 546-conjugated donkey anti-mouse IgG (Invitrogen), permeabilized with 0.5% Triton/PBS, re-fixed with methanol/acetone (50/50), stained with rabbit anti-γ-tubulin (Sigma), Alexa Fluor 647-conjugated donkey anti-rabbit IgG and Alexa Fluor 488-conjugated phalloidin, and mounted on slides using mounting medium containing DAPI (Prolong Gold, Invitrogen).
Images of single T cells on the EC apical surface were captured with a Leica TCS SP5 Spectral Confocal Microscope, 405UV using a 63X oil immersion objective and sequential scanning with 405 Diode, argon and He/Ne laser excitation lines of 405 nm, 488 nm, 543 nm, and 633 nm. Six Z slices were captured encompassing the entire T cell starting from the EC interface.

One hour after electroconvulsive stimulation (ECS), rats were deeply anesthetized with an overdose of pentobarbital solution, and removed brains were frozen in crushed dry ice and coronal sections of 14 μm thickness were cut on a cryostat and attached to a slide glass. Sections were fixed in 4 % paraformaldehyde and then incubated in PBS containing 4 % donkey serum (Chemicon, Temecula, CA) and 0.2 % Triton X-100 for 1 h at room temperature. Slices were incubated with rabbit c-fos antibody (1:500, sc-52, Santa Cruz Biotechnology, Inc, Santa Cruz, CA), and mouse anti-NeuN antibody (1:500, MAB377, Chemicon) overnight at 4 °C. After washing with PBS, the slices were incubated with Alexa Fluor 488-conjugated donkey anti-rabbit IgG (1:300, Molecular Probes, Eugene, OR), and Alexa Fluor 546-conjugated donkey anti-mouse IgG (1:300, Molecular Probes) for 2 h at room temperature. ProLong Gold Antifade Reagent (Molecular Probes) was used to protect samples from bleaching. Fluorescence images were obtained using the all-in-one fluorescence microscope, BZ-9000 (Keyence, Japan) through a 4× Plan-Apo objective (Nikon, Japan).

SH-SY5Y cells were seeded in 24-well plates (5 × 10³ cells/well) on poly-D-Lys-covered slides and treated with 1 µg/ml PFFs. The protocols have been previously described [39 (link), 44 (link), 45 (link)]. Primary antibodies recognized TH (Merck-Millipore), human α-SYN (Santa Cruz Biotechnology, Dallas, TX, USA) and α-SYN-pSer¹²⁹ (Abcam, Cambridge, UK). Secondary antibodies were as follows: Alexa Fluor 488 donkey anti-mouse, and Alexa 546 donkey anti-rabbit (1:500; Thermo Fisher Scientific, Waltham, MA, USA) and Alexa Fluor 546-conjugated donkey anti-mouse IgG (Molecular Probes, Eugene, OR, USA). Control sections were treated identically but omitting the primary antibody.