Anti guinea pig fitc

Anti-insulin antibodies were from Dako (Carpinteria, CA), anti-RyR2 from Affinity BioReagents (Golden, CO) or Millipore Corp. (Billerica, MA) and anti-calnexin from Sigma (St Louis, MO). The secondary antibodies used were anti-guinea pig FITC from Jackson Immuno Research (West Grove, PA). Alexa Fluor 635 anti-mouse IgG, and Alexa Fluor 635 anti-rabbit IgG, were both from Invitrogen (Eugene, OR). Antibodies against S-glutathionylated protein adducts were from Virogen Corp. (Watertown, MA).

Immunohistochemistry was performed as described below. Larval brains were dissected in PBS, and fixed in 4% formaldehyde/PBT solution at room temperature for 30–60 min. The brains were washed in PBT and blocked in 5–10% normal serum/PBT solution at room temperature for 30 min. Primary antibody reaction was performed in a solution containing primary antibodies and 1% normal serum in PBT at 4 °C overnight. The brains were washed in PBT. Secondary antibody reaction was performed in a solution containing secondary antibodies (1:200) and 1% normal serum in PBT at 4 °C overnight. The brains were washed in PBT and mounted in VECTASHIELD.
Primary antibodies: rabbit anti-Hth (1:1000; Adi Salzberg, Israel Institute of Technology, Israel), rat anti-Dpn (1:100; 11D1CH11, abcam), guinea pig anti-Lsc (1:1200), rat anti-Ncad (1:20; DSHB), mouse anti-Dscam1 (1:200; S. Lawrence Zipursky, UCLA, USA), mouse anti-Dig (1:200; 21H8, abcam) and rabbit anti-GFP Alexa488 conjugated (1:1000; Invitrogen A21311) antibodies. Secondary antibodies: anti-mouse Cy3, anti-mouse FITC, anti-mouse Cy5, anti-guinea pig Cy5, anti-guinea pig FITC, anti-rat Cy5, anti-chicken Cy3 (Jackson ImmunoResearch Laboratories) antibodies.
Confocal images were obtained by Zeiss LSM880 and processed using ZEN 2.3, ImageJ 1.52a and Adobe Photoshop CC 2019.