Alexa 568 anti mouse

Immunohistochemistry was done as described⁹¹ (link) except that the blocking step was overnight at 4°C. Primary antibodies: mouse anti-nc82 (DSHB, AB_2314866) 1:40, chicken anti-GFP (Abcam, ab13970) 1:1000, mouse anti-ChAT4B (DSHB, AB_528122), rabbit anti-GABA (Sigma, A2052). Secondary antibodies: Alexa-568 anti-mouse (Invitrogen) 1:400, Alexa-488 anti-chicken (Invitrogen) 1:400, Alexa-633 anti-mouse (Invitrogen) 1:400, Alexa-568 anti-rabbit (Invitrogen) 1:400.
Prolonged incubation (2-3 days at 4°C) with primary and secondary antibodies was required for homogeneous staining. Specimens were whole mounted in Vectashield (Vector Labs) on charged slides to avoid movement. Confocal stacks were acquired using a Zeiss 780 confocal microscope. Brains were imaged at 768 x 768 pixel resolution every 1 μm (0.46 x 0.46 x 1 μm) using an EC Plan-Neofluar 40x/1.30 oil objective and 0.6 zoom factor. All images were acquired at 16-bit color depth. Maximum projections of z stacks were made in Fiji.⁸⁹ (link)

Immunohistochemistry: anti-RBP2 (guinea pig polyclonal, 1:1000; kind gift of Eckart Gundelfinger; specifically recognizes RBP2 as verified by the knockout, [21 (link)]); anti-Cav1.3 (rabbit polyclonal, 1:500; Alomone Labs; [53 (link)]); anti-CtBP2/RIBEYE (mouse monoclonal, 1:200; BD Biosciences); Alexa488 anti-guinea pig (goat polyclonal; 1:500; Invitrogen), Alexa568 anti-mouse (goat polyclonal; 1.500; Invitrogen), Cy3 anti-rabbit (donkey polyclonal; 1:1500; Jackson Immuno Research); co-immunoprecipitation: affinity purified anti-RBP2-1318 (rabbit polyclonal; 1:100; directed against amino acids 261–284, NCBI reference sequence NP_001074857.1); anti-GFP (mouse monoclonal; 4 μl of 0.4 μg/μl; Roche); peroxidase-conjugated secondary antibodies: anti-rabbit (goat polyclonal; 1:20,000; Sigma-Aldrich); anti-mouse (goat polyclonal; 1:5000; Sigma-Aldrich); GST (glutathione-S-transferase) pull-down: anti-GAPDH (rabbit monoclonal; 1:1000; Cell Signaling Technology); anti-HA (mouse monoclonal κ16B12; 1:1000; Biozym); affinity-purified anti-Cav1.3α1_2022–2138 (rabbit polyclonal; 1:1000; [51 (link)]). Peroxidase-conjugated secondary antibodies: anti-mouse (polyclonal goat; 1:3000; Roth) and anti-rabbit (polyclonal goat; 1:3000; Roth).

Nervous systems of early third instar larvae were dissected in PB (100 mM NaH₂PO₄/Na₂HPO₄) pH 7.2, transferred to a polylysine-coated cover slip fixed with 4% formaldehyde in PB for 20 min at room temperature (RT) and rinsed in PBS plus 0.3% Triton X-100 (PBT) 3 × 15 min. Specimens were then incubated with anti-SCR 1/20 (Developmental Studies Hybridoma Bank, USA); chicken anti-GFP 1/2000 (Abcam) or rabbit anti-DCP- asp216 1/100 (Cell Signaling) in PBT overnight at 4°C in a wet chamber, washed in PBT 4 × 15 min, and incubated with secondary antibodies at 1/500 in PBT for 3 hr at RT: Alexa568 anti-Mouse (Invitrogen); CF633 anti-rabbit and Alexa488 anti-Chicken (Biotum). Secondary antibodies were washed 4 × 30 min in PBT and specimens were mounted in Vectashield (Vector Laboratories) between two aluminium-foil spacers, to avoid distortion of nerve cords, under number one cover glasses. Image stacks were captured on a Leica TCS-SP-5 confocal microscope.

Primary antibodies include: Cux1 (11733-1-AP, Proteintech), BRD9 (24785-1-AP, Proteintech), c-Jun (24909-1-AP, Proteintech), CBP (7389S, Cell Signaling), DYKDDDDK Tag (FLAG) (A00187S, Genscript), Total H3 (14269, Cell Signaling), H3K18Ac (13998, Cell Signaling), Anti-SV40 T antigen PAb419 (sc-58665, Santa Cruz), and Hsp70 (sc-7298, Santa Cruz). Primary antibodies against MCV LT (CM2B4, VP1, CM9B2) were gifted from Dr. Patrick Moore and Dr. Yuan Chang’s laboratory. 2T2 was gifted from Dr. Christopher Buck’s laboratory. Secondary antibodies for immunoblots include: goat anti-rabbit IRDye 800CW (926–32211, LI-COR), goat anti-mouse IRDye 800CW (926–32210, LI-COR), IRDye 800CW Streptavidin (926–32230, LI-COR). Secondary antibodies for immunofluorescence include: Alexa488 anti-mouse (Invitrogen), Alexa568 anti-mouse (Invitrogen), Alexa488 anti-rabbit (A11034, Invitrogen), Alexa568 anti-rabbit (A11036, Life Technologies).

Freshly isolated erythrocytes (directly after plasma separation) were smeared across glass slides and fixed with cold 4% paraformaldehyde for 30 min on ice. Erythrocytes were immunoblotted, after extensive washing, with an anti-SOD3 (sc-67089, Santa Cruz) and anti-CD235a (555569, BD) antibodies for all night at 4 °C, and subsequently washed and incubated with secondary antibodies Alexa-488 goat anti-rabbit and Alexa-568 anti-mouse (Invitrogen) for 1 h. The preparations were mounted in fluorescent counting medium (DAKO) and examined in a fluorescence microscope equipped with an ApoTome module for structured illumination (Zeiss AxioImager.z1, USA).
For flow cytometry analysis 40 μl of RBCs (5 × 10⁸ cells), collected directly after plasma separation, were fixed overnight in 1 mL of paraformaldehyde 4% at 4 °C followed by washing with staining buffer (PBS with 1% FBS, 0.09% NaN₃) and incubation with anti-SOD3 and with anti-CD235a antibodies for 30 min at room temperature. Samples were washed in PBS (with centrifugation at 200×g, 5 min) and incubated with anti-mouse or anti-rabbit Alexa Fluor-conjugated secondary antibodies for 20 min at RT. RBCs were suspended in staining buffer, data acquisition was performed with a FACSCantoII™ flow cytometer (BD Biosciences) and data analysis with FlowJo software (BD Biosciences).