Anti rat alexa fluor 546

The following antibodies and kits were used for immunohistochemistry and western blot: anti-mouse CD31, clone SZ31, Dianova; anti-human/mouse NG2, Millipore; anti-mouse E-cadherin, clone ECCD-2, Life Technologies; anti-human SMA, clone 1A4, Sigma-Aldrich; anti-human Ki67, Leica Biosystems; anti-mouse CC10, Abcam; anti-mouse proSP-C, Millipore; anti-human Hif-2α, Novus Biologicals; anti-mouse VEGF (A-20), Santa Cruz Biotechnology; anti-mouse actin, Santa Cruz Biotechnology; anti-rabbit HRP, DAKO; anti-rat Alexa Fluor 546, Molecular Probes; anti-rabbit Alexa Fluor 488, Molecular Probes; anti-rabbit FITC, Dianova; HRP-conjugated antibodies and ABC-kit, VECTOR Laboratories; DAPI, Sigma-Aldrich; haemalaun-eosin (HE) staining solution, Sigma-Aldrich; Trichrome Stain (Masson) Kit, Sigma-Aldrich; PAS Kit, Sigma-Aldrich.

Flies were reared at 25°C and aged for 5 days prior to dissection and staining as per Rideout et al. (2010) (link) with the following modifications: VNC samples were fixed in 4% PFA (40 mins at room temperature) immediately following dissection, to maintain tissue integrity and minimize cell loss. Samples were pre-incubated in 5% NGS overnight at 4°C. Samples were then incubated with primary antibodies for 3 days at 4°C, followed by an overnight incubation in secondary antibodies at 4°C. Primary antibodies used were: mouse mAb nC82 (1:50), mouse anti-AbdA (1:100, C-11), mouse anti-AbdB (1:10, 1A2E9-s), mouse anti-Acj6-s (1:20), mouse anti-AntP (1:20, 8C11-s), mouse anti-Ubx (1:20, FP3.38-s), rat anti-CadN (1:30, DN-Ex #8;) from DSHB, Univ. of Iowa. Additional primary antibodies used were chicken anti-GFP (1:1200, Abcam) and anti-AbdA (1:100, C-11 Santa Cruz Biotechnology). Secondary antibodies used included: anti-chicken Alexa Fluor488, anti-mouse Alexa Fluor633, anti-rat Alexa Fluor546, (1:300, Invitrogen Molecular Probes, Carlsbad, CA). Samples were left in 70% Glycerol/30% PBT overnight at 4°C prior to mounting with Vectashield (Vector Labs) and imaged with a Leica SP5 Microscope. Stacks of optical sections were generated at 0.5 µm intervals. Images were processed in Imaris 8.2.1 (Bitplane Scientific, AG, Zürich).

Formalin fixated paraffin embedded (FFPE) liver samples were cut into 5 µm strong sections and stained for CD11b (Abcam, Cambridge, UK, rabbit-anti-mouse) and Ly6G (Affimetrix-eBioscience, Frankfurt/Main, Germany, rat-anti-mouse). The primary antibody was used in a dilution of 1∶100 and a species specific secondary antibody with a HRP-conjugate was diluted 1∶500 to detect the primary antibody. Visualization was performed using 3,3′-diaminobenzidine tetrahydrochloride (DAB) (Sigma-Aldrich, Steinheim, Germany). Antigen retrieval was executed according to manufacturer's instruction. Nuclei were counterstained with Haematoxylin (Sigma-Aldrich, Steinheim, Germany). For each individual animal/genotype 7 pictures were taken in 100 and 200 fold magnification using an Olympus BX51. The 100 fold magnification was used for overview whereas the 200 fold magnification pictures were used for the detailed analysis and counting of the total CD11b⁺- or Ly6G⁺-cells per view field.
For the immunofluorescent staining of CD11b⁺-cells in the BDL-model, cryosections of liver tissue were made and stained with a CD11b-antibody (rat-anti-mouse, eBiosciences, Frankfurt/Main, Germany) used in a 1∶200 dilution. Visualization was performed using an anti-rat ALEXAFLUOR546 (Life Technologies, Darmstadt, Germany). Fluorescent microscopy pictures were taken by an AxioImager Z1 (Carl-Zeiss, Jena).