Alexa 555 conjugated donkey anti rabbit antibody

BmN-SWU1 cells were first seeded in a 24-well plate, and then transiently transfected or infected with BmNPV. At the indicated times, cells were fixed (4% paraformaldehyde, 15 min), permeabilized (1% Triton X-100 (Beyotime, Shanghai, China, ST677), 15 min), blocked (3% bovine serum albumin (BSA) and 10% sheep serum in PBS (blocking solution), 37 °C, 1 h), incubated with primary antibodies (1:200, 1.5 h, 37 °C), and incubated with secondary antibodies (1:500, 1 h, 37 °C) [41 (link)]. Triton X-100 (Beyotime, Shanghai, China, ST797), 4% paraformaldehyde (biosharp, Guangzhou, China, BL539A), BSA (Beyotime, Shanghai, China, ST023), sheep serum (ZSGB-BIO, ZLI-9021), primary antibodies (anti-Tubulin (Rabbit) (Beyotime, Shanghai, China, AF0001), anti-Brdu (mouse) (Roche, Mannheim, Germany, 11444611001), anti-Flag (mouse) (Abmart, Shanghai, China, M20008H), and anti-HA (Rabbit) (Invitrogen, Shanghai, China, 71–5500)), second antibodies (Alexa 555-conjugated donkey anti-rabbit antibody (Invitrogen, Shanghai, China, A32732), and Alexa 488-conjugated donkey anti-mouse antibody (Invitrogen, Shanghai, China, A32723)) were used in the present study.

Vibratome sections (80 µm) from the brains of either adult wildtype mice or Mdga1 KO mice (Ishikawa et al., 2011 (link)) (a gift of T. Yamamoto, Kagawa University, Japan) were permeabilized at room temperature (RT) for 40 min in PBS containing 0.5% Triton X-100 (Sigma-Aldrich). Sections were then blocked overnight at 4 °C in PBS containing 10% normal horse serum (NHS), 0.5% Triton X-100, 0.5 M glycine (Sigma-Aldrich, #G8898), 0.2% gelatin (Sigma-Aldrich, #G7041). Sections were then washed in PBS-0.5% Triton X-100 at RT and incubated at 4 °C for 48 hr with MDGA1 antiserum (dilution 1:500) in PBS containing 5% NHS, 0.5% Triton X-100% and 0.2% gelatin. Afterwards, sections were washed in PBS-0.5%Triton X-100 at RT before overnight incubation at 4 °C with Alexa555-conjugated donkey-anti-rabbit antibody (Invitrogen, #A32794) in PBS containing 5% NHS, 0.5% Triton X-100% and 0.2% gelatin. Before mounting coverslips with Mowiol-4–88 (Sigma-Aldrich), sections were washed in PBS-0.5% Triton X-100. Images were acquired using a Leica SP8 confocal microscope (Leica Microsystems).