Donkey anti rabbit igg conjugated to alexa fluor 488

Immunostaining of fixed MDBK cells in 24-well plates infected with LSDV-SODis-p67HA-BLV-Gag at MOI 0.05 was performed as previously described at two days post-infection (30 (link), 38 (link)). Live staining of HeLa cells infected with virus at MOI 0.05 in 4-well Permanox^® chamber slides (Thermo Fisher Scientific, USA) coated with poly-L-lysine (P8920, Sigma, USA) was performed at two days post infection as previously described (30 (link), 38 (link)) using 1:100 anti-p67 antibody. Donkey anti-rabbit-IgG conjugated to Alexa Fluor 488 (Life Technologies, USA) was used as the secondary antibody at 1:1000.

Rats were anesthetized with 10% chloral hydrate (0.3 ml/100 g, i.p.) and perfused transcardially with 200 ml of 0.9% saline followed by 300 ml of 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). The brain tissues were removed and postfixed in 4% paraformaldehyde overnight and cryoprotected in 30% sucrose. Thirty-micron-thick frozen sections from the rat brains were cut using a freezing microtome and serially collected throughout the hippocampus. Free-floating tissue sections were rinsed three times with PBS-T. The tissue sections were incubated with 10% normal donkey serum in PBS-T for 2 h, followed by incubation with rabbit anti-p-NF-κB p65 (1:500, Cell Signaling Technology, Inc., Beverly, MA) and mouse anti-glial fibrillary acidic protein (GFAP) monoclonal antibody (1:600, Abcam, Cambridge, UK) at 4 °C for 24 h. After three 5-min rinses in PBS, the sections were incubated with donkey anti-rabbit IgG conjugated to Alexa Fluor® 488 and donkey anti-mouse IgG conjugated to Alexa Fluor® 594 (1:500, Life Technologies, Carlsbad, CA, USA) in the dark for 2 h at 37 °C. Fluorescence intensity was visualized under a confocal microscope (FV1000, Olympus Corp., Tokyo, Japan). The intensity on four slides (three to four sections per slide) was averaged for each animal and then normalized by that of the control group.

Mouse monoclonal antibodies against the peroxisomal membrane protein PMP70 (Sigma, St. Louis, MO), HIV-1 p24 (Abcam, Cambridge, MA), GFP (Abcam), myc (Millipore Sigma, Etobicoke, ON, Canada), and beta-actin (Abcam) were purchased from the indicated suppliers. Rabbit polyclonal antibodies to PEX7, PEX11B, PEX13, PEX19, catalase, beta-catenin, and AU1 were obtained from Abcam; rabbit polyclonal antibody to PEX2 (PXMP3) was purchased from Pierce (Rockford, IL); rabbit monoclonal antibody to β-TrCP (D12C8) was purchased from New England Biolabs (Whitby, ON, Canada); and rabbit polyclonal antibody to the tripeptide SKL was produced as previously described (61 (link)).
Donkey anti-mouse IgG conjugated to Alexa Fluor 680, goat anti-rabbit IgG conjugated to Alexa Fluor 800, donkey anti-mouse IgG conjugated to Alexa Fluor 488, donkey anti-rabbit IgG conjugated to Alexa Fluor 488, donkey anti-rabbit IgG conjugated to Alexa Fluor 546, and donkey anti-mouse IgG conjugated to Alexa Fluor 546 were purchased from Invitrogen.