Goat anti rabbit cy3

Antibody staining was performed by standard procedures for imaginal discs [66 (link)]. Rabbit anti-Cleaved Caspase-3 (1:400, Cell Signaling Technology, CST, Cat # 9661, Danvers, MA, USA) was used as a primary antibody, and goat anti-rabbit CY3 (1:1000, Life technologies, Cat # A10520) was used as a secondary antibody.

All tissues used for immunohistochemistry for histopathology were immersion fixed in 4 % paraformaldehyde. Tissue used for sections was embedded in OCT media. 7 μm sections were obtained and either stained with hematoxylin and eosin or processed for immunohistochemistry. Primary antibodies used include rabbit anti-Iba1 (Wako Chem, Osaka, Japan), CD3 (Rabbit anti-mouse CD3, Abcam, Cambridge, MA) and CD19 (rat anti-mouse CD19, Abcam). Secondary antibodies used were either goat anti-rabbit Cy3 or goat anti-rat Alexa 488 diluted 1:300 (both life technologies). It was also necessary to enhance the endogenous signal of DsRed⁺ lymphocytes through immunohistochemical approaches. Sections were incubated with the primary antibody mouse-anti DsRed (St. Cruz Biotechnologies, Dallas, TX) or rabbit-anti red fluorescent protein (Abcam) and counterstained with DAPI (Sigma, St. Louis, MO) to facilitate orientation.
Retinal whole mounts were preserved in 4 % paraformaldehyde. Retinas where preincubated in 0.3 % Triton X-100/PBS (Sigma) for 4 h and blocked in 0.1 % BSA/0.3 % Triton X-100/PBS for 1 h. Primary antibodies were diluted 1:300 in 0.3 % Triton X-100/PBS and incubated for 18 h at 4 °C. After extensive washing in PBS retinas were incubated with the secondary antibodies (1:200 in PBS) for three hours. Retinal wholemounts were coverslipped and images were taken on an Olympus BX41 microscope.

Immunofluorescence staining was performed on frozen graft tissue sections after 10-min fixation with ice-cold acetone and/or methanol (7 μm thickness). Sections were blocked for 1 hour at room temperature (RT) with 3% fetal bovine serum in phosphate buffered saline before incubation with primary antibodies against hC7 LH7.2 (Sigma-Aldrich) in a 1:500 dilution (Supplementary Table S1 online), desmoglein-1 (Fitzerald Industries, Acton, MA), involucrin (Sigma-Aldrich), keratin 10 (in-house), complex IV subunit II MTCO2 (Abcam, Cambridge, UK) overnight at 4 °C. Secondary antibody incubation with Alexa Fluor goat antimouse 488 (Invitrogen, Paisley, UK), goat antirabbit Cy3 (Life Technologies, Paisley, UK), and strep 488 was followed for 1 hour at RT. Sections were stained with 4'.6-diamidino-2-phenylindole (5 mg/ml) and mounted using a ProLong Gold antifade agent (Life Technologies). These were also stained by a hematoxylin and eosin histochemical technique. Staining was visualized and imaged using a Leica DMLB upright microscope (Leica Microsystems CMS, Wetzlar, Germany) and a Zeiss Axiophot 2 (Zeiss, Oberkochen, Germany) and processed using Image-Pro 6.2 (MediaCybernetics, Rockville, MD). Confocal imaging was carried out on a Zeiss LSM 510 Meta laser confocal microscope (Zeiss). Postprocessing was carried out using ImageJ.